THE REACTION MECHANISM OF HUMAN ADENYLATE KINASE. 

THE STEADY-STATE KINETICS OF THE MUTANTS CONSTRUCTED BY PROTEIN ENGINEERING OF SITE-DIRECTED MUTAGENESIS

 

 

Takanori Ayabe 1, 2 and Minoru Hamada 2, *

 

1 The Second Department of Surgery, 2 Department of Hygiene, Miyazaki Medical College, Miyazaki, 889-1692, Japan.

 

* Correspondence should be addressed to Minoru HAMADA

 

Address: Department of Hygiene, Miyazaki Medical College, Kiyotake, Miyazaki, 889-1692, Japan.

E-mail: mhamada@post1.miyazaki-med.ac.jp

Phone: +81-985-85-0873

FAX: +81-985-85-5177

 

Abbreviations: ADP, adenosine 5'-diphosphate; AK, adenylate kinase; AKc, chicken adenylate kinase; AMP, adenosine 5'-monophosphate; ATP adenosine

5'-triphosphate; EDTA, ethylenediaminetetraacetate; hAK1, human adenylate kinase 1; NADH, nicotinamide adenine dinucleotide; NMR; nuclear magnetic resonance; Tris, 2-amino-2-(hydroxymethyl)-1,3-propanediol; WTAK, wild-type adenylate kinase.

 

Running titile:  THE REACTION MECHANISM OF HUMAN ADENYLATE KINASE

 

 

 

 

 

 

 

 

 

CONTENTS

 

I.     INTRODUCTION

II.     THE METHODS: CONSTRUCTION OF THE PMEX8-HAK1 EXPRESSION VECTOR.  RANDOM SITE-DIRECTED MUTAGENESIS AND PROTEIN      ENGINEERING

III.    LYSINE RESIDUES IN HUMAN ADENYLATE KINASE ARE ESSENTIAL   FOR INTERACTION WITH ADENINE NUCLEOTIDES

IV.    SITE-DIRECTED MUTAGENESIS OF THREONINE RESIDUES IN THE N     TERMINAL REGION OF HUMAN ADENYLATE KINASE

V.     THE REACTION MECHANISM AFTER HYDROPHOBIC ALTERATIONS       IN THE C-TERMINAL α-HELIX OF HUMAN ADENYLATE KINASE

VI.    PERSPECTIVES AND FUTURE WORK

 

 

 

 

 

I.  INTRODUCTION

 

      The isolation of rabbit muscle myokinase (ATP-AMP transphosphorylase, adenylate kinase, myokinase, EC 2.7.4.3.) in crystalline form was first described by Noda and Kuby [1,2], who also determined several of its physicochemical properties [3].  Adenylate kinase is a ubiquitous enzyme [4, 5] and it has since been prepared in either crystalline or homogeneous form and characterized from a variety of sources, including rabbit [1-3, 6], porcine [7], carp [8], bovine liver mitochondria [9], rat muscle [10], rat liver [10, 11, 12], porcine liver [13], rat hepatomas [14], rat brain [15], human erythrocytes [16], bakers' yeast [17], calf muscle and liver [6], human liver mitochondria [18], porcine heart [12], human muscle [19, 20] and liver [20], normal human serum and an aberrant form in human dystrophic serum [21], human FSH (Fascio-scapulo-humeral type) dystrophic muscle and liver [22], bovine heart mitochondria [23], and Escherichia coli [24].

      Adenylate kinase is involved in the interconversion of adenine nucleotides in the cells.  In vertebrates, three isozymes (AK1, AK2, and AK3) have so far been characterized [25].  AK1 is present in the cytosol of skeletal muscle, brain, and erythrocyte [5, 26], while AK2 is found in the mitochondrial intermembrane space of the liver, kidney, spleen, and heart [26].  These isozymes catalyze the reaction: MgATP + AMP フ MgADP + ADP, and contribute to homeostasis of the adenine nucleotide composition in the cell [27].  In cellular metabolism, it is considered that adenylate kinase provides AMP to rate-limiting steps in energy- generating pathway (e.g., glycolysis) as an allosteric activator, resulting in stimulation of the rate of the pathway. AK3, which catalyzes the reaction: MgGTP + AMP フ MgGDP + ADP, is present in the mitochondrial matrix of the liver and heart [28].  GTP is generated by substrate level phosphorylation in mitochondria; therefore, AK3 is thought to be involved in transferring the phosphate moiety from the guanine nucleotide pool to the adenine nucleotide pool together with nucleotide diphosphate kinase [29].  By gene structures of the three isozymes by Nakazawa et al. [25], the comparison of amino acid sequences and the genomic structure of the three isozymes revealed that a segment corresponding to either exon 5 of the AK2 gene or a part of exon 3 of the AK3 gene is missing in the AK1 gene.  Phylogenic analysis suggested that AK1, a shorter molecule, would have been separated from a longer molecule very early in evolution of adenylate kinase.  AK2 and AK3 would have diverged along the longer-molecule lineage.

      In human genetic disorders, an aberrant AK isozyme has been specifically found in the serum of Duchenne dystrophic patients [21].  An aberrant adenylate kinase isozyme was detected [30] and isolated from the serum of a patient with Duchenne Muscular Dystrophy [21], which resembled the isolated human liver mitochondrial adenylate kinase [18, 31] in its kinetics, stability, and electrophoretic properties [32], but by immunological detection it appeared structurally to be a muscle type [31, 32].  It would be of interest from both a genetic and a pathogenetic standpoint to understand precisely which covalent alterations may have taken place in this human variant adenylate kinase isozyme and whether or not it represents a fetal species.  An AK1 deficiency associated with hemolytic anemia has been reported [33, 34, 35], and the replacement of Arg-128 with Trp in AK1 was considered to be the cause of the enzyme deficiency. The same mutant prepared by site-directed mutagenesis resulted in reduced catalytic activity and decreased solubility [34].  Based on these findings, it seems reasonable to study the properties of various mutant AKs in order to better understand disorders due to such mutantations. 

      The amino acid sequences have been deduced for the porcine muscle adenylate kinase [31, 32], for one species of human muscle adenylate kinase designated AK1 [36], for calf and rabbit muscle adenylate kinase [37], for chicken muscle adenylate kinase [38], for adult bovine heart mitochondria designated AK2 [39, 40], and from E. coli [41, 42].  The X-ray crystal structures of forms A and B of the porcine adenylate kinase [43, 44, 45] and of the yeast cytosolic adenylate kinase [46] and of the E. coli enzyme [47] were elucidated, and the human, porcine, and rabbit muscle adenylate kinases were studied by 1H nuclear magnetic resonance spectroscopy [48, 49, 50, 51].

      The amino acid analyses were presented [20] for the crystalline normal human liver, calf liver, and rabbit liver adenylate kinases, and compared with the normal human muscle, calf muscle, and rabbit muscle adenylate kinases.  The liver-types as a group and the muscle-types as a group show a great deal of homology, but some distinct differences are evident between the liver and muscle enzyme groups, especially in the number of residues of His, Pro, and half-cystine and in the presence of Trp in the liver enzymes [20].  Porcine AK1 and adult bovine heart AK2, display some degree of homology especially near the amino terminus [23, 40].  The adk gene encoding adenylate kinase in E. coli was cloned in pBR322, and the primary structure of the E. coli adenylate kinase was deduced from the nucleotide sequence of the adk gene [42].  Gilles et al. [52] then showed that a thermosensitive adenylate kinase was due to the substitution of a serine residue for a proline residue at position-87; they also reported a circular dichroism study of both species [53].  In the case of the chick, a cDNA clone for muscle adenylate kinase was isolated from a cDNA library of chick skeletal muscle poly (A)+RNA, and the DNA sequence was determined by Kishi et al. [38], who thereby deduced its primary structure.

      The MgATP-binding site of rabbit muscle adenylate kinase was located by combining the NMR data [50] with the X-ray diffraction data [43, 44, 45]. (Fig. 1, Fig. 2)  It was near three protein segments, five to seven amino acids in length, which are homologous in sequence to segments found in other nucleotide-binding phosphotransferases, such as myosin and F1-ATPase, ras P21 and transducin GTPases, and cAMP-dependent and src protein kinases, suggesting that equivalent mechanistic roles of these segments exist in all of these proteins [54].  Table 1 shows sequence homologies among the ATP-binding region of adenylate kinase and the segments of other proteins.  The AMP-binding site was also located by NMR studies [55], and a final deduced structure for the ternary complex was presented.  A detailed analysis of the steady-state kinetic reaction mechanism was presented earlier by Hamada et al. [56] and together with substrate binding data on the muscle-type adenylate kinases and derived peptide fragments [57, 58], some mechanistic ideas surfaced, especially in pinpointing the two sites for binding of MgATP/MgADP and of AMP/ADP.

      AK has ten α-helices and five β-strands [59].  The primary structures of some isoenzymes of AK have been well identified, but their tertiary structures have not been completely elucidated  [60].  Extensive attempts have been made to identify the substrate binding sites of AK: by X-ray crystallography of porcine AKs [44, 61] and yeast AK [46], by NMR studies of isolated rabbit muscle AK [50, 62, 63] and the synthetic MgATP2- binding fragments of AK [56], and by site-directed mutagenesis studies of chicken AK (AKc) [64, 65, 66, 67, 68, 69] and human AK (hAK1) [70].  However, the active site for catalysis and the important residues for substrate-binding have not been exactly determined.  To elucidate the AK structural model proposed by X-ray crystallography and NMR studies, interaction with substrates and specific residues should be tested by site-directed mutagenesis and steady-state kinetics.  In hAK1, the conserved arginine residues (R44, R97, R132, and R138) were substituted with alanine and the ATP site in the original X-ray model was reassigned to the AMP site (Fig. 3); these arginine residues were determined to be essential for catalytic activity [70].  The reassignment of the AMP site in the revised X-ray model has also been suggested by NMR data [71] and by the data for the F86W mutant of AKe [72].  However, a detailed analysis of MgATP2- and AMP2- sites remains to be done in an AK structural model.

      To elucidate the minimum requirement of amino acid residues for the active center in human adenylate kinase (hAK1), we carried out random site-directed mutagenesis of key lysine residues (K9, K21, K27, K31, K63, K131, and K194), threonine residues in the N-terminal region (T35 and T39), and hydrophobic residues (V182, V186, C187, L190, and L193), which were conserved in mammalian AK1 species, with the pMEX8-hAK1 plasmid [73].  To define a structural AK model previously proposed by Kim et al [70], we constructed the pMEX8-hAK1 plasmid [73].  We partially modified the method for random site directed mutagenesis based on the phosphorothioate technique [74, 75, 76].

 

 

II.   THE METHODS:  CONSTRUCTION OF THE PMEX8-HAK1 EXPRESSION VECTOR.   RANDOM SITE-DIRECTED MUTAGENESIS AND PROTEIN ENGINEERING

 

1.  OUTLINE

      The pMEX8-hAK1 vector [73] was devised from the pAK plasmid [77], which could directly express human adenylate kinase proteins without recombination and its single strand DNA could be withdrawn with a helper phage for random site directed mutagenesis.  The conserved key residues were engineered to obtain mutants for kinetic analysis.  This pMEX8-hAK1 will be a powerful tool for site directed mutagenesis to detect the substrate-enzyme interaction for human adenylate kinase including various other enzymes.  

 

2.  BACKGROUND     

        Kim et al. [77] chemically synthesized an artificial gene (Fig. 4), expressed for the human cytosolic adenylate kinase (hAK1), and obtained kinetic parameters of mutants on replacement of conserved arginine residues with alanine.  From in vitro mutagenesis studies [70], the binding site for AMP was predicted and revised from X-ray crystallographic studies [78].  A new hAK1 expression vector was constructed from a pAK plasmid [77] to permit effective site-directed random mutagenesis.  We partially modified the method for random site-directed mutagenesis based on the phosphorothioate technique [74, 75, 76].

 

3.  MATERIALS AND METHODS

3.1.  Materials. 

      JM101/pAK (JM101 transformed with plasmid pAK ligated with human cytosolic adenylate kinase cDNA) was kindly provided by Dr. Kim at Hang-Yang University and Dr. Uesugi at Yokohama National University Institute of Technology [77].  The plasmid pMEX8 vector was the product of MoBiTec (Gottingen).  Bacterial strain, JM109, was purchased from TaKaRa Shuzo, Co., Ltd. (Tokyo).  A bacterial strain TG1 and a site-directed mutagenesis kit, the Sculptor in vitro mutagenesis system, were purchased from Amersham LIFE SCIENCE.  All other reagents were of the highest purity commercially available.

 

3.2.  Construction of human cytosolic adenylate kinase vector (pMEX8-hAK1).

      Fig. 5 shows the construction of the pMEX8-hAK1 expression vector.  JM101/pAK was cultured in LB medium (5 ml) containing ampicillin (50 μg/ml), and the plasmid pAK ligated with human cytosolic adenylate kinase cDNA [70] was purified.  The plasmid pAK was digested with Cla I and Sal I and a pMEX8 vector with EcoR I and Sal I.  The digested hAK1 gene and the pMEX8 plasmid were subjected to agarose gel electrophoresis and extraction with DE81 paper.  The digested pMEX8 vector with EcoR I and Sal I was dephosphorylated with calf intestine alkaline phosphatase (TaKaRa Shuzo, Co., Ltd.)  The hAK1 gene was ligated into the pMEX8 vector with a DNA ligation kit (TaKaRa).  By this ligation step, Sal I sites of both hAK1 and pMEX8 could be ligated.  However, the EcoR I site of pMEX8 and the Cla I site of hAK1 were still not ligated.  These uncohesive ends were blunted with a blunting kit (TaKaRa) and then ligated as described above.  Competent cells, JM109, were transformed by the ligated vector pMEX8 with hAK1.  After incubation with Hi-competent broth (Wako Co., Tokyo), transformed cells were cultured on LB plates containing ampicillin.  Each single colony obtained was then cultured in LB medium (5 ml) containing ampicillin (50 μg/ml) at 37 ℃ overnight with shaking.  After purification of plasmid DNA from each cultured clone, EcoR I and Sal I digestion was performed to confirm the insertion of hAK1 by 5% PAGE.  Plasmid pMEX8 ligated with the hAK1 gene was analyzed by DNA sequencing.  All molecular biological experiments were carried out according to the procedures in the textbook [79].

 

3.4.  Purification of single strand pMEX8-hAK1 DNA.

      A single colony of JM109/pMEX8-hAK1 (JM109 transformed with pMEX8 hAK1) was cultured in 10 ml of TYP medium (1.6% Tryptone, 1.6% yeast extract, 0.5% NaCl, 0.25% K2HPO4) containing 50 μg/ml of ampicillin at 37℃ overnight until the A600 was approximately 0.5.  Helper phages (VCS-M13, Stratagene, La Jolla, CA, U.S.A.) were added to the culture medium at a multiplicity of infection between 10 and 20 (a phage : cell ratio of between 10 : 1 and 20 : 1) with 25 μg/ml of kanamycin.  The medium was cultured at 37℃ with vigorous aeration overnight and was centrifuged at 3,000 X g for 20 min.  To the supernatant was added a solution of polyethyleneglycol 6000 and 2.5 M NaCl.  The mixture was allowed to stand at room temperature for 15 min.  After centrifugation of the solution, the pellet was resuspended with TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0), and single stranded DNA of pMEX8-hAK1 was extracted with TE-saturated phenol and chloroform according to the manufacturer's recommendations. 

 

3.5. Random site-directed mutagenesis of hAK1.

      The antisense primers were 5'-CGAAGATGATYXXAGTCTTCTTAAGC-3' for Lys9 residue, 5'-GCACTGGGTACCYXXGCCAGAACC-3' for Lys21, 5'- GCACGATYXXCTCGCACTGGG -3' for Lys27, 5'-GTGTAGCCGTAYXXCTGCACGATTTTC-3' for Lys31, 5'-CCAGCTGACCYXXTTCCATGATTTC-3' for Lys63, 5'-GTTTCGCCGCGYXXCAGCAGGCG-3' for Lys131, 5'-CGAAGATGATYXXAGTCTTCTTAAGC-3' for Lys194.  5'-GTAGACAGGTGYXXGTAGCCGTATTTC-3' for Thr35,  5'- GCAGGTCACCYXXAGACAGGTG-3' for Thr39,  5'-CCTGAGAGAAYXXTTCGTCAAC-3' for Val182, 5'- GCAGGAGAGTGCAYXXCTGAGAGAATACTTC -3' for Val186, 5'-CCAGGTGAGTYXXTACCTGAGAG-3' for Cys187,  5'-TCAGAGCTCYXXGTGAGTGCATA-3' for Leu190, and 5'-CGTCGACTATTATTTYXXAGCGTCCAGGTG-3' for Leu193. 

      These primers were synthesized with a DNA synthesizer (Applied Biosystems, Model 394) and phosphorylated with T4 Polynucleotide Kinase (WAKO Co., Tokyo).  The underlined letters, YXX, represent a target codon for random mutagenesis, where X was either A, G, C or T, while Y was either G or C.  Site directed mutagenesis was carried out with a combination of the SculptorTM in vitro Mutagenesis Kit (Amersham LIFE SCIENCE, Buckinghamshire, England) using these site-specific primers annealed to the template single strand DNA of pMEX8 hAK1.  Fig. 6 shows a flow chart on site-directed mutagenesis.  This method was based on the phosphorothioate technique [74, 75, 76], that is, dCTP was used instead of dCTPαS during both the annealing and extension reaction of the randomly constructed oligonucleotides.

 

3.6.  Screening of mutants by DNA cycle sequencing.

      A homoduplex mutant DNA constructed by site-directed mutagenesis was transformed with competent cells (TG1) prepared according to the directions in the mutagenesis kit and was spread on an LB plate containing 50 μg/ml of ampicillin.  Single clones were cultured in 10 ml of LB medium overnight.  A double-stranded DNA of the plasmid was purified according to the manufacturer's instructions (Flex Prep Purification Kit, Pharmacia Biotech, Tokyo, Japan).  The mutant plasmid DNA was sequenced by the dideoxy method [78].  Sequencing primers labeled with fluorescent isothiocyanate were the product of Japan Bioservices.  The DNA sequence of the forward primer was 5'-TGGAATTGTGAGCGGATAAC-3', and that of the reverse primer was 5'-AAAATCTTCTCTCATCCGCC-3'.  Polymerase chain reaction (PCR) was performed with the AmpliCycleTM Sequencing kit (Perkin Elmer, Branchburg, NJ, U.S.A.) with a DNA Thermal Cycler (Model PJ 480, Perkin Elmer Cetus) according to the partially modified protocol described as follows.  To enhance Taq DNA polymerase activity, AmpliTaqTM DNA Polymerase solution and Taq DNA Polymerase (Promega, Madison, WI, U.S.A) were mixed at a ratio of 9 : 1 as a cycling mixture.  Eight μl of a master mixture was made by mixing 2.8 μg of double-stranded DNA, 1 μl of dimethylsulfoxide (Sigma Chemical Co., St. Louis, MO, U.S.A.), and 2 pmol of the FITC-labeled sequence primer.  This mixture was incubated at 95℃ for 10 min and immediately cooled on ice.  Two μl of the cycling mixture and 0.2 unit of a Perfect Match Enhancer (Stratagene, La Jolla, CA, U.S.A.) were added to the cooled master mixture.  PCR conditions were initial denaturation at 95℃ for 5 min, 20 cycles at 95℃ for 30 s, 53℃ for 30 s, and 72℃ for 60 s, and 20 cycles at 95℃ for 30 s and 72℃ for 60 s.  PCR products were denatured by 95% formamide.  Electrophoresis of the PCR product was performed by an autosequencer (Shimadzu, DSQ1, Kyoto, Japan).  DNA sequencing of the mutant plasmid was performed with forward and backward sequencing primers to avoid undesirable mutations in the entire hAK1 gene.  Fig. 7 shows a DNA ladder pattern of Lys194-mutants.  The position at 195 in the C terminal α-helix of the mutant was confirmed to be a stop codon (TAATAG).

 

3.7.  Expression and purification of wild type AK (WTAK) and mutant AK.

      JM109/pMEX8-hAK1 (wild type AK) and TG1/pMEX8-mutant-hAK1 were separately cultured in 10 ml of LB medium containing 50 μg/ml of ampicillin overnight at 37℃ and were transferred into 250 ml of LB medium.  After 1 hr culture, isopropyl-β-D-thio-galactopyranoside (IPTG) was added at a final concentration of 1 mM, and growth in the medium was continued for 16 hr under the same conditions.  E. coli cells were centrifuged at 5,000 X g for 20 min, and the pellet was suspended in 10 ml of a standard buffer [20 mM Tris-HCl, 1 mM EDTA, 0.1 mM dithiothreitol (DTT), pH 7.4].  All chromatographic steps were carried out at 4℃ in the chromatochamber.  The suspension of E. coli cells was disrupted with an ultrasonicator (Model 250 sonifier, Branson Ultrasonics Co., Danbury, CT, U.S.A.) at 20 kHz and 20 W for 3 min on ice.  The homogenate was centrifuged at 12,000 X g for 20 min at 4℃.  The supernatant was subjected to affinity chromatography on Blue Sepharose CL-6B (φ1 X 5 cm) which had been equilibrated with the standard buffer and eluted with a NaCl gradient (0 M to 1 M NaCl in the standard buffer) at a velocity of 0.5 ml/min.  AK protein was determined by 12.5% polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate (SDS-PAGE) to confirm a single AK protein [70], and the AK fraction was concentrated by centrifuging with Centriplus-10 (Amicon, Inc., Tokyo, Japan).  The concentrated AK (0.5 ml) sample was loaded onto a Superose 12 column (φ 1 X 30 cm) and eluted with imidazole buffer (5 mM imidazole-HCl, 1 mM EDTA, 0.1 mM DTT, pH 6.9) at a velocity of 0.5 ml/min.  Each fraction was also determined by 12.5% SDS-PAGE to confirm a single band of AK protein (Fig. 8).

 

3.8.  Kinetic analysis of the forward reaction of AK.

      Enzyme activity was assayed in the forward direction by adding various amounts of MgSO4, ATP, and AMP.  Fig. 9 shows the method of AK assay.  The initial velocity of the forward reaction was measured by observing the absorbance change at 340 nm with a Cary 2290 spectrophotometer (Varian, Mulgrave, Australia) [for NADH, in a coupled-enzyme assay in the presence of pyruvate kinase and lactate dehydrogenase to monitor ADP formation at 25℃ as previously described] [56].  The forward reaction mixture in a total of 1 ml contained 75 mM triethanolamine hydrochloride (pH 7.4), 120 mM KCl, 0.2 mM NADH, 0.3 mM PEP, 0.3 mg/ml bovine serum albumin (BSA), 10 units of LDH, 5 units of PK, and 1.0 mM MgSO4, and the various concentrations of MgATP2- and AMP2- were as follows: five combinations selected from 0.05, 0.1, 0.2, 0.5, 1.0, 2.0, 3.0, 5.0 mM MgATP2- and at a fixed concentration of 2 mM AMP2- were used in the determination of the apparent Michaelis constants (Km) for MgATP2-.  Five combinations selected from 0.05, 0.1, 0.2, 0.5, 1.0, 2.0, 3.0, 5.0 mM AMP2- and a fixed concentration of 2.0 mM MgATP2- were used in the determination of the apparent Km for AMP2-.  The reaction was initiated by the addition of 10 ml of the recombinant hAK1 solution diluted to the desired concentrations.  The AK sample was diluted with a buffer (5 mM imidazole-HCl, 1 mM EDTA, 0.1 mM DTT, 1% BSA, pH 7.4).  Km and Vmax were estimated using a double-reciprocal plot [80], and kcat was calculated by dividing Vmax by the total amount of enzyme (Et) present in the reaction mixture.  One unit of enzyme activity is defined as the amount of enzyme that produces 1 μmol of ADP in one minute.      

 

3.9.  Others.

      The concentration of protein was determined by the method of Lowry [81].  Concentrations of adenine nucleotides and NADH were spectrophotometrically determined using millimolar extinction coefficients of 15.4 and 6.22, respectively.

 

 

III.   LYSINE RESIDUES IN HUMAN ADENYLATE KINASE ARE ESSENTIAL FOR INTERACTION WITH ADENINE NUCLEOTIDES

 

1. OUTLINE

      To elucidate the minimum requirement of amino acid residues for the active center in human adenylate kinase (hAK1), we carried out random site-directed mutagenesis of key lysine residues (K9, K21, K27, K31, K63, K131, and K194), which were conserved in mammalian AK1 species, with the pMEX8-hAK1 plasmid [82].  Twenty different mutants were obtained and analyzed by steady-state kinetics, and all mutants showed an activity loss by Km and/or kcat effects on one or both of MgATP2- and AMP2-.  The results have led to the following conclusions.  (1) Lys9 would appear to interact with both MgATP2- and AMP2- but to a larger extent than with AMP2-. (2) Lys21 is likely to play a role in substrate-binding of both MgATP2- and AMP2- but more strongly affects MgATP2-.  (3) Lys27 and Lys131 would appear to play a functional role in catalysis by interacting strongly with MgATP2-.  (4) Lys31 would appear to interact with MgATP2- and AMP2- at the MgATP2- site.  (5) Lys63 would be more likely to interact with MgATP2- than with AMP2-. (6) Lys194 in the flanking C-terminal α-helix would appear to interact not only with MgATP2- but also with AMP2- at the MgATP2- site by stabilizing substrate-binding.  The loss of the positively charged ε-amino group of lysine affects both the affinity for the substrate and the catalytic efficiency.  Hence, hydrophilic lysine residues in hAK1 would appear to be essential for substrate enzyme interaction with the coordination of the some arginine residues, reported previously [70].

 

2.  BACKGROUND

      To evaluate a mutant form of hAK1 as a model of enzyme deficiency disorders, and secondarily, to elucidate the minimum requirement of amino acid residues at the active center in hAK1, by subsequent analysis of Arg-mutants in hAK1 [70], we substituted the lysine residues conserved in mammalian AK, which were suggested to be essential to interact with the substrate by NMR and X-ray diffraction analyses [50, 61, 63, 66, 83, 84].  As reported previously, Lys21, Lys27, and Lys194 were suggested to interact with the substrates [85].  The reason why we replaced lysine is that the changes from an aliphatic positively charged and hydrophilic basic amino acid would be largely changed compared to those from other amino acids with a neutral, hydrophobic, and non-charged side chain.  To define a structural AK model previously proposed by Kim et al. [70], we constructed the pMEX8-hAK1 plasmid [73].  We partially modified the method for random site-directed mutagenesis based on the phosphorothioate technique [74, 75, 76].  To elucidate the function of the positive ε-amino groups of the hydrophilic lysine residues, we mutated Lys9, Lys21, Lys27, and Lys31 in the head region, Lys63 and Lys131 in the middle region, and Lys194 in the tail region of hAK1.  These mutants were analyzed by steady-state kinetics. 

 

3.  RESULTS

3.1.  Site-directed mutagenesis of Lys residues and purification of wild type AK (WTAK) and mutant AKs.

      The results of site-directed random mutagenesis of pMEX8-hAK1 are summarized in Table 2.  Twenty-six mutants at seven target residues were determined with backward and forward sequencing primers, and the efficiency of the random mutagenesis was averaged to 27.7% (26 mutants/94 colonies). 

 

3.2.  Kinetic parameters of mutant AKs.      

      Steady-state kinetic data for WTAK and each mutant in the forward reaction are shown in Table 3.

 

(A) Properties of K9-mutants. 

      The Km values for K9P were slightly decreased (0.2-fold) for MgATP2- and AMP2- compared to those of WTAK; however, the decrease in kcat  was comparable (9.3%) among the other K9-mutants.  The kcat of K9F was decreased to 0.3% with an unchanged Km for both substrates.  With K9L there was a 6.8-fold increase in Km for AMP2-, kcat  was decreased to 0.4% and the kcat/Km value for AMP2- was dramatically decreased to 6.5 X 10-2%.  The Km values of K9T showed more than 10-fold increases for MgATP2- and AMP2-, and the kcat was reduced to 1.6%.  These results strongly suggest that K9-mutants interact with both MgATP2- and AMP2- and have a larger effect on AMP2- than on MgATP2-. 

      K9-mutants affected the affinity for both MgATP2- and AMP2- but more strongly interacted with AMP2-, which decreased the catalytic efficiency.  Lys9 would be essential for catalysis by interacting with MgATP2- and AMP2-.  The glycine-rich loop plays an important role in catalysis [63, 83, 86]. 

 

(B) Properties of K21-mutant.

      The Km values of K21P for MgATP2- and AMP2- were markedly increased, 19.4-fold and 13.8-fold, respectively.  The kcat  value was decreased to 5.0%.  The results for K21P suggested interaction with both MgATP2- and AMP2-; however, the mutant more strongly affected MgATP2-.

      The K21P could make the flexible P-loop bend in any direction, which could affect the substrate-binding site, reducing the affinity for both substrates and decreasing the kcat value.  The Lys21 residue would be essential for catalysis and substrate-binding.  Lys21 was proposed to be close to the MgATP binding site [50], and the ε-amino group was suggested to be close to the subsite for the

γ-phosphate of ATP [64].  According to an NMR study of K21M in AKc, Lys21 may play a key structural role [66].  Based on NMR analyses of K21R and K21A, Lys21 would orient the triphosphate chain of MgATP to the proper conformation required for catalysis [84]. 

 

(C) Properties of K27-mutants. 

      The Km values for AMP2- of K27R, K27L, and K27I mutants were not changed (1.1- to 1.5-fold), while those for MgATP2- were moderately increased (3.6- to 8.9-fold).  The kcat  values were decreased greater than 2.0%, and the kcat/Km values were significantly decreased.  The K27-mutants appeared to interact with MgATP2- to a greater extent than with AMP2- and showed correspondingly decreased catalytic efficiency.  It is surprising that for K27R, although the positive charge was conserved, a decrease in kcat (to 1.7%) was observed despite an unchanged Km.

      K27-mutants showed moderately decreased affinity for MgATP2- and decreased catalytic efficiency.  From the data we obtained, in fact, Lys27 would appear to play a functional role in catalysis at the MgATP2- site.  The K27R mutant, in spite of the change of an ε-amino group to a guanidino group retaining a positive charge, showed a largely reduced kcat.  The catalytic function of Lys27 could not be replaced by a conservative residue arginine, and the ε-amino group was presumed to be necessary for phosphoryl binding and transfer.  Based on NMR studies of K27M in AKc [66], Lys27 was nonessential for either catalysis (including substrate binding) or structure; however, the discrepancies between this report and our data might be due in part to the difference in experimental conditions using different species and types of mutations in the side chains.  The roles of these same mutations might differ between AKc and hAK1.  This should be tested using the K27M mutant in hAK1. 

 

(D) Properties of K31-mutants.

      The Km values of K31I were unchanged for MgATP2- and AMP2-, 0.9- and 1.4-fold, respectively, while the kcat  value decreased to 1.9%.  K31F showed an increased Km for MgATP2- (12.1-fold), and the kcat/Km values for MgATP2- were decreased to 9.5 X 10-2%  In contrast, the Km value of K31S dramatically increased (23.6-fold) for AMP2-, and the kcat/Km for AMP2- showed a large decrease to 2.3 X 10-2%.  K31I did not significantly affect the affinity for either substrate, although K31F strongly affected MgATP2- and K31S greatly affected AMP2-.  K31-mutants appeared to interact with MgATP2- and AMP2- and to be essential for catalysis.

      A study of K31-mutants revealed that K31F showed a decreased affinity for MgATP2- but K31S showed reduced affinity for AMP2-.  Lys31 was proposed to be at or near bound MgATP2- [63]; however, the result affected both MgATP2- and AMP2-.  When the enzyme forms a ternary complex by substrate-binding, the side chain of Lys31 at the MgATP2- site might permit the AMP2- substrate to interact with the phosphate of AMP2- for catalysis.  Lys63 is denoted on the right side close to the AMP2- site (Fig. 10). 

 

(E) Properties of K63-mutant. 

      The Km values of K63F increased 8.1-fold for MgATP2- and were unchanged (0.8-fold) for AMP2-.  The kcat decreased to 0.8%, and the kcat/Km value for MgATP2- was decreased to 9.5 X 10-2%.  The K63-mutant interacted with MgATP2- and showed decreased catalytic efficiency.

      K63F showed a greatly reduced affinity for MgATP2- and decreased catalytic efficiency.  By photoaffinity labeling analysis, the amino acid domain (Gly64 to Arg77) was proposed to constitute the neighborhood identifiable with AMP binding during binary-to-ternary complex formation [87].  When two substrates (MgATP2- and AMP2-) bind with the enzyme and form a ternary complex, some conformational change could occur which allows the side chain of Lys63 access to the MgATP2- substrate. 

 

(F) Properties of K131-mutants. 

      Results for K131A showed a dramatic increase in Km for MgATP2- (16.9 fold), a decrease by 0.4% for kcat  and a large decrease in kcat/Km for MgATP2- (2.6 X 10-2%).  The Km of K131F showed a small change (3.2-fold for MgATP2- and 1.6-fold for AMP2-), but the kcat values were decreased to 0.4%.  K131 mutants were suggested to interact greatly with MgATP2- in catalysis.

      The 120-133 helical region exists at the top of the AK model (Fig. 10).  K131 mutants showed moderately decreased affinity for MgATP2- and decreased catalytic efficiency.  Based on kinetic analyses of R132A in hAK1 [70], Arg132 next to Lys131 would appear to be essential for catalysis by interacting strongly with MgATP2- and only partially with AMP2-.  According to NMR studies of R132M in AKc, Arg132 was important for transition-state stabilization [68].  The residue 131 to 141 segment was observed to move in substrate holding, which correlates especially with ATP-binding [88].  Lys131 would be essential for catalysis by interacting with MgATP2-.   

 

(G) Properties of K194-mutants.

      The Km value of K194S for MgATP2- was moderately increased (14.5-fold), while that for AMP2- was not changed (3.4-fold), and the kcat  was not significantly changed to 37.3%.  In the case of K194L, the Km showed a moderate increase of 9.6-fold for MgATP2- but decreased 0.1-fold for AMP2-, and the kcat slightly decreased to 13.7%.  The kcat/Km of K194L for MgATP2- markedly decreased to 1.4%; however, that for AMP2- strikingly increased the catalytic efficiency for AMP2- (229.4%).  On the other hand, despite the fact that the K194V mutant was similar to K194L, the Km value of K194V was greatly increased (20.8-fold) for AMP2- and unchanged (0.9-fold) for MgATP2-.  The kcat  decreased to 1.4%.  The kcat/Km of K194V for AMP2- was markedly decreased (7.1 X 10-2%) in spite of a small decrease in catalytic efficiency for MgATP2- (1.5%).  K194I showed a small decrease of 0.2-fold in Km values for MgATP2- and 0.6-fold for AMP2-, and the kcat was decreased to 2.4%.  K194P showed the largest decrease (0.8%) in kcat among all the K194-mutants.  K194-mutants interacted not only with MgATP2- but also with AMP2-.

      The flanking C-terminal α-helix is denoted on the left side of the proposed AK model, and Lys194 is in the tail section (Fig. 10).  AKc consists of 193 amino acid residues [37], and hAK1 consists of an additional lysine at the 194 position.  The Leu190 residue in AKc appears to participate in the components of the MgATP-binding domain [67].  In hAK1, K194S showed a moderately decreased affinity for MgATP2-.  However, similar mutations in K194V and K194L affected the affinity for MgATP2- or AMP2- differently and decreased the catalytic efficiency.  Different hydrophobic changes such as valine and leucine at position 194 may affect the binding of MgATP2- and AMP2- substrates, and the mutants may cause a conformational change in the C-terminal α-helix by altering its hydrophobicity.  The C-terminal α-helix might appear to move and to access and interact with the AMP2- substrate forming a ternary complex.  Lys194 would appear to interact not only with MgATP2- but also with AMP2-, possibly indirectly, and thus the flanking C-terminal α-helix would be essential for catalysis by stabilizing the transition state and holding the substrates; Lys194 could then not be replaced by other amino acids.

 

4.  DISCUSSION

      X-ray structural studies indicate that AK protein has ten α-helices and five β strands [59] and that the active center cleft opens to some extent (Fig. 10).  In a previous study of hAK1, replacement of the arginine residues at R44, R97, R132, and R138 with alanine resulted in decreased catalytic efficiency [70].  The loss of the positively charged guanidino groups of the arginine residues would inhibit catalytic efficiency, and the arginine residue was suggested to be essential for catalysis by interacting with the negative charge of the phosphates of adenine nucleotides.  In hAK1, site-directed mutagenesis has not been performed except for Arg-residues.  In the present study, to elucidate the minimum requirement for amino acid residues at the active center in hAK1 and to elucidate the interaction with substrates and specific residues, we substituted the positively charged ε-amino groups of Lys9, Lys21, Lys27, Lys31, Lys63, Lys131, and Lys194 residues by random site-directed mutagenesis.  These lysine residues were well conserved in mammalian species. 

      We have not analyzed the secondary structures of all mutants with circular dichroism spectra because these lysine residues might not be directly involved in the secondary structures, as reported in a previous paper; in the case of replacement of arginine with alanine, very large differences were not displayed [70].  However, as a control for the Lys-mutant, circular dichroism studies should be shortly performed on several kinds of Lys-mutants.  K27V and K131P mutants could not be analyzed because they were insoluble in the standard buffer described in "Materials and Methods", which cause might be considered due to the helical change observed in other proteins.  The conformation or hydrophobicity of these insoluble mutants might have been altered upon folding; however, because they were not solubilized, these mutants should be detected by optical rotational dichroism (ORD) studies.  In human genetic disorders associated with nonspherocytic hemolytic anemia, an AK1 deficiency in erythrocytes has been reported [33, 34, 35].  The nucleotide sequence of the patient's AK1 gene resulted in a replacement of Arg-128 with Trp, and the properties of the mutant AK expressed in vitro showed reduced catalytic activity as well as decreased solubility in AKc [34].  A new mutant enzyme constructed by site-directed mutagenesis may be useful for studying the pathophysiological mechanism of disorders due to a mutant enzyme derived from a point mutation.  The affinity for substrates changed and/or the catalytic efficiency of Lys-mutants were variously decreased and in spite of a decreased solubility, which event could explain a part of the cause of hemolytic anemia derived from the AK1-mutant.

      In a brief summary, the steady-state kinetic studies above have led to the following conclusions.  (i) Lys9 would be essential for catalysis by interacting with both MgATP2- and AMP2- but by affecting AMP2- more strongly. (ii) Lys21 would be essential for catalysis by interacting with both MgATP2- and AMP2- but by affecting MgATP2- more strongly.  (iii) Lys27 and Lys131 would appear to play a functional role in catalysis by interacting strongly with MgATP2-.  (iv) Lys31 would appear to interact with MgATP2- and AMP2- at the MgATP2- site and be essential for catalysis.  (v) Lys63 is likely to interact to a much greater extent with MgATP2-.  (vi) Lys194 in the flanking C-terminal α-helix would interact not only with MgATP2- but also with AMP2- at the MgATP2- site by holding both substrates.  The loss of the ε-amino group of lysine (Lys9, Lys21, Lys27, Lys31, Lys63, Lys131, Lys194) resulted in decreased or unchanged affinity for the substrates and reduced catalytic efficiency.  The positive charge of lysine is not replaced in nature and may interact with the negatively charged phosphates of MgATP2- and AMP2-, which in turn may orient the phosphate chains of the two substrates to the proper conformation required for catalysis. 

        We would predict insertion of important lysine residues as shown in a new AK structural model (Fig. 10).  These lysine residues would take part in a phosphoryl transfer reaction between MgATP2- and AMP2-, in cooperation with the other active site residues, as in the case of arginine residues [70].

 

 

IV.  SITE-DIRECTED RANDOM MUTAGENESIS OF THREONINE RESIDUES IN THE N-TERMINAL REGION OF HUMAN ADENYLATE KINASE

 

1.  OUTLINE

        To elucidate the catalytic and structural roles of threonine residues in human AK, the Thr35 and Thr39 mutants were analyzed by steady-state kinetics.  The Km values of T35P and T35Y were not changed for MgATP2- and AMP2-, and the kcat values were decreased by 2.5% compared to those of wild-type AK (WTAK).  Thr35 was suggested to be essential for catalysis.  The Km values of T39S, T39V, and T39P were increased 5.6- to 59.0-fold for AMP2-; however, the kcat values were not reduced.  Although the Km values of T39F and T39L were unchanged, the kcat values were reduced by more than 1.8%.  Thr39 appears to play an important role in the binding of AMP2- and to be essential for catalysis.  As noted above, a hydroxyl group of the Thr residue in human AK appears to be important.

 

2.  BACKGROUND

      In human AK, the Arg and Lys residues have been substituted and the resultant mutants were analyzed by steady-state kinetics [70, 73].  However, Thr residues in human AK have not been analyzed and the substrate-binding regions for MgATP2- and AMP2- have not been sufficiently evaluated.  In this study, to elucidate the physiological role of Thr residues in the N-terminal region of human AK, random site-directed mutagenesis of Thr35 and Thr39 was performed with pMEX8-hAK1 plasmid [73].  Thr35 and Thr39 are conserved in the AK family and are located in the β-strand in the N-terminal region [63].  Although the structural and functional roles of the Thr35 residue are not known, the Thr39 residue in yeast AK (AKy) was suggested to be in proximity to the adenine moiety of adenosine-B in the AKy - MgAP5A complex [46].  On the basis of results of T39A in AKc, Thr39 was not indicated to be essential to binding or catalysis [65].  We report kinetic data on Thr mutants constructed by site-directed mutagenesis of human AK. 

 

3.  RESULTS

3.1.  Random site-directed mutagenesis of Thr35 and Thr39 residues.

      After random site-directed mutagenesis of the Thr35 residue, T35P (CCC) and T35Y (TAC) mutants were confirmed by DNA sequencing of twelve colonies.  Five T39-mutants, T39S (TCC), T39F (TTC), T39L (TTG), T39P (CCC), and T39V (GTC), were obtained by screening of sixteen colonies (Table 4).  The efficiency of mutagenesis was 25% (7 mutants/28 colonies).

 

3.2.  Kinetic properties of the reaction mechanism of the mutant enzymes.

      Steady-state kinetic results in the forward reaction are shown in Table 5.

 

(A) Steady-state kinetics of T35-mutants.

      The Km value for MgATP2- of the T35P mutant was not significantly increased (2.1-fold), while that for AMP2- was slightly decreased 0.2-fold compared to the results for WTAK.  The kcat and Km/kcat (MgATP2-) values for T35P were significantly decreased to 2.5 and 1.2%, respectively.  The Km values of T35Y were not significantly changed (3.2-fold for MgATP2- and 1.4-fold for AMP2-) but the kcat value was greatly decreased by 0.4%.  The Km/kcat value for MgATP2- of T35Y was significantly decreased by 0.1%.  T35 mutations did not affect the affinity for either substrate but did reduce catalytic efficiency.

 

(B) Steady-state kinetics of T39-mutants.

      The Km value for MgATP2- of T39V was not significantly increased (2.7 fold), while that for AMP2- was greatly increased (13.3-fold), and the kcat value was unchanged compared to that of WTAK.  In the case of the T39P mutant, the Km value for MgATP2- was unchanged, 0.6-fold; however, that for AMP2- was significantly increased by 58.9-fold, and the kcat value was not changed compared to that of WTAK.  On the other hand, the Km value of T39S was not significantly increased (2.5-fold), for MgATP2-, but that for AMP2- was slightly increased (5.6 fold), and the kcat value was decreased by 2.8%.  The Km values of T39F were not greatly changed (3.2-fold for MgATP2- and 0.3-fold for AMP2-) and the kcat  value was greatly decreased by 0.7%.  The Km values for T39L were not changed, 1.9 fold for MgATP2- and 0.4-fold for AMP2-, and the kcat  value was strongly decreased by 1.8%.  T39-mutants affected, the affinity for AMP2- much more strongly than for MgATP2-, which indicated a reduction in catalytic efficiency.

 

4.  DISCUSSION

     Thr35 and Thr39 residues are conserved in AK families and located in the β strand in the N-terminal region of human AK.  In an earlier study of structural and functional roles of the Thr39 residue, Thr39 in AKy was suggested to be in proximity to the adenine moiety of adenosine-B in the AKy - MgAP5A complex [46].  However, based on the results of T39A in AKc, Thr39 was not essential to binding or catalysis [65].  This discrepancy indicates that Thr39 might play a different role among species, that different experimental procedures for X-ray diffraction analyses and NMR studies might provide differing interpretations.  In human AK, to elucidate the physiological function of Thr35 and Thr39 residues, we performed random site-directed mutagenesis with pMEX8-hAK1 plasmid [73].

      By analysis of steady-state kinetics on T35-mutants, both T35P and T35Y showed decreased catalytic efficiency without affecting the affinity for both MgATP2- and AMP2-.  Although the Thr35 residue is denoted in the deep cleft of the AK protein model (Fig. 11), even a Pro mutation in the deep β-strand cleft might not affect the binding regions of the substrates.  Thr35 might possibly indirectly participate in catalysis.  Replacements of threonine by proline and tyrosine are considered to be a hydrophobic and aromatic change, and the resultant decreased hydrophilicity may reduce catalytic efficiency.  A hydroxyl group in the side chain of Thr35 might stabilize the phosphoryl transfer between MgATP2- and AMP2- substrates.  In an earlier paper, in porcine AK (AKp), the His36 residue was suggested to be located in the binding site of ATP and to be involved in catalysis [45, 89, 90].  However, in AKc, by NMR study on His36-mutants [66], His36 was suggested not to be directly involved in catalysis but to stabilize the ternary structure.  This discrepancy might suggest that His36 plays a different role in AKp and AKc.  In human AK, Thr35 next to His36 appears to play an important role in catalysis.

      From the results of steady-state kinetic studies of Thr39 mutants, T39V and T39P more significantly decreased the affinity for the AMP2- substrate than for MgATP2-, but did not affect catalytic efficiency.  From the above two mutants, Thr39 appears to play an important role in the binding of AMP2-.  Thr39 exists in the stretch of theβ-strand (residues 35-40) [63], and the replacement of threonine by proline at position 39 might bend this β-strand in any direction; thus a conformational change which might strongly affect the binding region of AMP2-.  On the other hand, T39V and T39L mutants had similar properties and structures; however, T39L decreased the catalytic efficiency without affecting the affinity for the MgATP2- and AMP2- substrates, even though T39V strongly affected the affinity for the AMP2- substrate.  The difference in hydrophobicity of the side chain of valine and leucine mutations at position 39 might affect the binding of the AMP2- substrate and inhibit phosphoryl transfer indirectly.  The T39S mutant affected the binding of the AMP2- substrate and decreased catalytic efficiency.  In contrast, T39F did not affect the affinity for either AMP2- or MgATP2- but decreased catalytic efficiency.  Although T39F showed a kinetic perturbation similar to that of T35Y, that is, there was a decrease in kcat without change in Km, a change from a hydroxyl group to an aromatic group decreased the catalytic efficiency without affecting substrate binding.  A loss of the hydroxyl group in the side chain of threonine may cause a hydrophilic change, an alteration which might indirectly inhibit catalysis.  From the above-noted kinetics, Thr39 was suggested to play an important role in the binding of AMP2- substrate and phosphoryl transfer between MgATP2- and AMP2- indirectly.  The hydroxyl group of threonine might be important for the binding of the AMP2- substrate and for catalysis. 

      In the previous study, the synthetic peptide corresponding to residues 32-40 of rabbit muscle enzyme was revealed to bind MgεATP2- [85].  By an X-ray diffraction study of AKy crystallized as a 1:1 complex with the inhibitor P1, P5 di(adenosine-5'-)pentaphosphate [46], Thr39 was found to be very close to the adenine moiety of MgATP (the adenosine-B site).  However, by an NMR study of the T39A mutant in AKc [65], Thr39 was suggested not to be functionally essential, and the detailed interaction between AMP and AKc was somewhat different from that of the depicted X-ray structure of AKy.  Through our kinetic analysis of Thr mutants in human AK, Thr35 might be essential for catalysis, and Thr39 appears to play an important role in the binding of AMP2- and in catalysis.  A loss of the hydroxyl group in the side chain of Thr residue in human AK appears to affect catalysis and substrate binding.  To elucidate the ternary complex of the two substrates and each mutant AK, NMR or detailed X-ray crystallographic studies must be performed in the future.  Our next effort will be directed toward detailed analysis and comparison of other Thr and Ser residues in the C-terminal α-helix of human AK and to more accurately define the assignments for the interactions between AK and two substrates.

 

 

 

 

V.     THE REACTION MECHANISM AFTER HYDROPHOBIC ALTERATIONS      IN THE C-TERMINAL α-HELIX OF HUMAN ADENYLATE KINASE

 

1. OUTLINE

      To elucidate if the C-terminal region in human adenylate kinase participates in interaction with the substrate (MgATP2- and/or AMP2-), hydrophobic residues (Val182, Val186, Cys187, Leu190, and Leu193) were substituted by site-directed mutagenesis and fifteen mutants were analyzed by steady-state kinetics.  The change in the hydrophobic residues in the C-terminal α-helix affects the affinity for substrates (Km), that is, not for MgATP2- but for AMP2-, and the catalytic efficiency (kcat).  The results obtained have led to the following conclusions: (1) Val182 may interact with both MgATP2- and AMP2- substrates but interacts to a greater extent with MgATP2-.  (2) Val186 appears to play a functional role in catalysis by interacting with both MgATP2- and AMP2- to nearly the same extent.  (3) Cys187 appears to play a functional role in catalysis.  (4) Leu190 appears to interact with both MgATP2- and AMP2- substrates but to a greater extent with AMP2-.  (5) Leu193 appears to interact with both MgATP2- and AMP2- but to a greater extent with AMP2-.  The activity of all mutants decreased due to changing substrate-affinity.  The nearer the residue located in the C-terminal end, the mutation affected not only MgATP2- but also AMP2- substrate binding.  The hydrophobic alterations have disrupted hydrophobic interactions with substrates, which might have destabilized the conformation of the active site.  The C-terminal α-helix of human adenylate kinase appears to be essential for playing a functional role in catalysis by interacting with adenine substrates.

 

2. BACKGROUND

      In the present study we evaluated the flanking C-terminal α-helix of this enzyme, by substitution of the hydrophobic residues conserved in mammalian AK, which have not been examined for interactions with the adenine nucleotide substrates.  We previously reported that Lys194 at the C-terminus of human AK interacted with both MgATP2- and AMP2- for substrate binding [82], even though the C-terminal region was located in the MgATP2- binding site [70].  To elucidate the participation of the C-terminal α-helix in the interaction with MgATP2- and AMP2-, we selected the hydrophobic residues which might affect the affinity for the adenine nucleotides.  We mutated Val182, Val186, Cys187, Leu190, and Leu193 residues by site-directed mutagenesis; and the various mutants produced were analyzed by steady-state kinetics. 

 

3.  RESULTS

3.1.  Site-directed mutagenesis and purification of mutant adenylate kinases

      The results of site-directed random mutagenesis of pMEX8-hAK1 are summarized in Table 6.  Nineteen mutants at five target residues were constructed with backward and forward sequencing primers.  For the Leu193-mutant series, the TAG mutation at position 193 was confirmed, that is, the deletion of residues 193 194 produced the unique L193Stop mutant.  The mutant pMEX8-hAK1 plasmids were expressed, and the various AK enzymes produced were purified to homogeneity by column chromatography using Blue Sepharose and Superose 12.  These AK mutants possessed the same chromatographic elution patterns as the wild type enzyme.  However, four mutants, Val186N, Leu190P, Leu190T, and Leu190N could not be purified after disruption of the expressed cells due to their insolubility in the standard buffer described in Methods.  This probably resulted from changes in solubility due to the specific mutations in the C-terminal α-helix.

 

3.2. Kinetic parameters of mutant adenylate kinases 

      Steady-state kinetic data for wild type AK and each mutant in the forward reaction are shown in Table 7.  The Km, kcat, and kcat/Km values obtained for each mutant were compared to the wild-type AK and expressed as the mutant/WTAK ratio (Table 7).

 

(A) Properties of the V182-mutants.

      The Km values of V182A for MgATP2- and AMP2- were markedly increased (by 11-fold and 12-fold) compared to wild-type AK.  The kcat value for this mutant was drastically decreased (to 0.1% of the control.)  The kcat/Km ratios for the two nucleotide substrates were dramatically decreased (to 6.2 × 10-3 and 5.7 × 10-3% of the control,  respectively.)  The Km values for V182G showed a 7.4-fold increase for MgATP2- and a 1.9- fold increase for AMP2-; the kcat value was reduced to 0.7% of the control.  Km values for V182S were elevated (by 2.0- and 1.5-fold) for MgATP2- and AMP2-, respectively; the kcat value was greatly decreased (to 0.5% of the control).  In general, the three V182 mutants suppressed the interaction with both the MgATP2- and the AMP2- binding sites, but to a lesser extent with AMP2-.  The catalytic efficiency (kc) for each was very low (i.e., the enzymatic activity was barely detectable.)

 

(B) Properties of the V186-mutants.

      The Km values for V186S increased by 7.0- and 7.5-fold for MgATP2- and AMP2-, respectively; the kcat value decreased to 1.0% of the control.  The Km values for V186G were elevated by 1.3- to 1.9-fold for MgATP2- and AMP2-, respectively, while the kcat value decreased to 1.2% of control.  These results strongly suggest that V186-mutants interact with both the MgATP2- and AMP2- binding sites to nearly the same extent and that they both have a profound effect on catalytic efficiency.

     

(C) Properties of the C187-mutant.

      The Km values for C187V were decreased slightly for MgATP2- (by 0.6-fold) and increased slightly (by 1.9-fold) for AMP2-, compared to those for wild type AK.  However, the kcat value was greatly decreased (to 0.2% of the control).  These results strongly suggest that the C187V mutant has a relatively large effect on catalytic efficiency but has little effect on substrate binding. 

 

(D) Properties of the L190-mutants.

      Km values for L190A were increased for both substrates, but to a lesser extent for MgATP2- than for AMP2- (i.e., 2.7-fold vs. 5.3-fold, respectively); the kcat decreased to a value only 1.6% that of the control.  The L190S mutant showed a markedly increased Km value for both MgATP2- (26-fold) and AMP2- (82-fold); the kcat value was decreased to 0.5% of the control and the kcat/Km ratios were decreased to 2.0 × 10-2% for MgATP2- and to 6.5 × 10-3% for AMP2-, relative to the wild type.  The L190A and L190S mutants both affected the affinity for MgATP2- and AMP2-, but the increase in Km was much greater for AMP2- in the case of L190S (82-fold) than for the L190A mutant (5.3-fold).  The effects on Km were greater for L190S for both AMP2- and MgATP2- binding sites.  The effects of MgATP2- on Km were greater for L190S (26-fold) than for L190A (2.7-fold), a pattern consistent with the results for AMP2-.  It appears, therefore, that residue L190 is essential for substrate binding. 

 

(E) Properties of the L193-mutants.

      The Km values for L193I increased by 7.8-fold for MgATP2- and 32-fold for AMP2-; the kcat value decreased to 0.7% of the control.  Thus, the L193I mutant strongly affects the affinity for AMP2- and to a lesser extent the affinity for MgATP2-.  The Km values of L193Q showed an increase for MgATP2-.  The Km values for L193Q increased by 9.3-fold for MgATP2- and by 7.4-fold for ATP2-; the kcat value was decreased to 0.4% of the control.  There was a large decrease in the kcat/Km ratio for both substrates (to 4.8 × 10-2% for MgATP2- and 5.9 × 10 2% for AMP2-).  The L193Q mutant affects the affinity for both substrates and decreases profoundly the catalytic efficiency.  The Km values for the L193P and L193S mutants showed a small elevation (2.7-fold and 1.6-fold for MgATP2-, respectively) and a decrease to 0.1-fold for AMP2- for both mutants; the kcat values were decreased to 13.5% and 6.4% of the control for L193P and L193S, respectively.  The kcat/Km ratio for L193P for AMP2- was increased to 153% of the control, while that for L193S was decreased, yielding a catalytic efficiency for AMP2- of 54% of the control; the values of kcat/Km (MgATP2-) were nearly identical (see Table 7) for both mutants.  The Km values of L193F and L193R for MgATP2- were relatively unchanged (0.4- to 1.0-fold), while that for AMP2- was marginally increased (3.2- to 3.8-fold); the kcat values for these two mutants were 2.2% and 1.6% of the control.  In the case of L193Stop, the Km value showed a moderate increase of 11-fold for AMP2- but was increased by only 1.6-fold for MgATP2-.  The kcat value decreased to 2.2% and 1.6% of the control for L193F and L193S, respectively.  The deletion mutation (L193Stop), in which residues 193 and 194 are deleted from the C-terminal α-helix, showed an increase in Km that was much greater for AMP2- than for MgATP2- (11-fold vs. 1.6-fold, respectively).  A similar result was obtained in the case of the L193I mutant in which the values of Km for AMP2- and MgATP2- were increased by 32- and 7.8 fold, respectively (see Table 7).  The above results on seven L193 mutations strongly suggest that these mutants interact with both substrate binding sites but more strongly with AMP2- than with MgATP2-.

 

4.  DISCUSSION

      In a previous study of human AK1, lysine residues were suggested to be essential for catalysis by interacting with the negative charges of the phosphate groups on the two adenine nucleotide substrates.  However, the detailed analysis of the C-terminal α-helix of human adenylate kinase has not yet been reported.  In this study five residues (Val182, Val186, Cys187, Leu190, and Leu193) were targeted for mutation in the C-terminal α-helix.  These residues are depicted on the left side of the wild type hAK1 model shown in Fig. 12.  X-ray crystallographic studies indicate that the AK protein has ten α-helices and five β-strands [59] and that the active center cleft opens to some extent on substrate binding (Fig. 12).  Adenylate kinase is predicted to undergo large domain movements upon binding to substrate [88].  The adenosine binding center is generated by a hydrophobic pocket which consists of a β-sheet structure, the loop of residues 16-22, and the α-helix between the segments of residues 23-30 and 179-194.  In this C-terminal region, hydrophobic residues have been well conserved, and we investigated the participation of binding in this region.

      Our results for the steady-state kinetics analysis of mutants for five residues in the C-terminal α-helix indicate that Val182 might be located close to the MgATP2- site, compared to Val186, Cys187, Leu190, and Leu193 residues (Fig. 12).  It appears that the greater the interaction with the C-terminal α-helix, the more the Km values are increased for AMP2-, and to a lesser extent for MgATP2-.  Leu193Stop, the deletion-mutant of residues 193-194,  results in a shortened C terminal-α-helix.  This mutant AK enzyme showed an 11-fold increase in Km for AMP2- and very little change for MgATP2-; kcat decreased substantially, relative to the control (Table 7).  The Leu193Stop mutant affects enzymatic activity and could affect affinity for AMP2-, as well.  Val182, Val186, Cys187, Leu190, and Leu193 residues constitute the hydrophobicity in the C-terminal α-helix.  Mutations in this region lead to loss of hydrophobicity, which leads to an increase in Km and decreased activity of this enzyme by changing the conformation of the C-terminal α-helix.  These alterations have affected the α-helix of residue16-23 and residue131-139, which might have changed the Km values for the two substrates.  The flanking C-terminal region would appear to interact with either or both substrates, as was suggested by the results of Ayabe et al. [82], who showed that C terminal mutants at position Lys194 affected not only the affinity for MgATP2- but for AMP2-, as well.  In the present study, the steady-state kinetic results with various mutant species of wild type hAK1 have led to the following conclusions: (1) Val182 likely plays a role in substrate-binding for both MgATP2- and AMP2-, but the interaction is stronger for MgATP2-; (2) Val186 appears to play a functional role in catalysis by interacting with both MgATP2- and AMP2- to nearly the same extent; (3) Cys187 would appear to play a functional role in catalysis; (4) Leu190 appears to interact with both MgATP2- and AMP2- but to a greater extent with AMP2-; (5) Leu193 appears to interact not only with MgATP2- but also with AMP2- but to a greater extent with AMP2-.  The nearer the residue is located to the C-terminal end, the mutation affects not only MgATP2- but also AMP2- substrate binding.  Results from mutations in the C-terminal region of chicken AK1 showed that Km values for Leu190 mutants were affected to a greater extent for MgATP2- than for AMP2- [67].  The kinetic data for the two chicken AK1 mutants (L190K and the deletion mutant at positions 190-193) are fully in accord with our results on human AK1.  The C-terminal α-helix may be involved in interaction with MgATP2- [82].

 

5.  SUMMARY 

      In the present study, the mutation of hydrophobic residues in the C-terminal α-helix resulted in decreased or unchanged affinity for the two substrates and reduced catalytic efficiency.  The loss of hydrophobicity in the C-terminal α-helix may lead to reduced binding of MgATP2- and AMP2-, because this α-helix may align the phosphate groups in the two substrates to the proper conformation required for catalysis.  This C-terminal mutation decreased the hydrophobic degree for MgATP2-, which alteration might have indirectly affected the AMP2- binding site, or which mutation affected the surrounding residue 16-23 and the α-helix residue 131-139; as a result, this changed the binding of MgATP2- and AMP2- and the phosphoryl transfer reaction.  We predict that movement of the C-terminal α helix in solution covers both substrates during the enzymatic reaction.  Based on the X-ray crystallographic structures, the AMP binding domain was predicted to undergo a movement of 8Å upon AMP binding, and the ATP binding domain was to move up to 30 Å upon binding of ATP [88, 91].  To elucidate the structure of the ternary complex of the substrates with mutant AK enzymes, detailed NMR studies or X-ray crystallographic studies would be required.  Circular dichroism and optical rotational dichroism studies must also be performed to better understand the changes resulting from our mutations in the hydrophobic C-terminal α-helix.  For the future, detailed analyses of our human AK1 mutants should provide a conformational model that can be used to clarify structure-function relationships for this enzyme.  The important role of the flanking C-terminal α-helix of adenylate kinase in catalysis may be further elucidated by X-ray crystallographic studies with substrate analogs and/or by NMR spectroscopy studies in solution.  Such studies should advance our understanding of the catalytic mechanism of this important kinase.

 

 

VI. PERSPECTIVES AND FUTURE WORK

 

      The AK structural model proposed by X-ray crystallographic studies is obtained in a crystal with substrate-inhibitory analogs, the structural data from NMR studies are confirmed in solution.  A new model based on the present data by steady-state kinetics (Fig. 3, 10, 11, 12) might be more likely for a dynamic and physiological model in solution than X-ray crystallographic and NMR studies indicate.  These discrepancies would be expected to be solved in the near future.  To elucidate the structures of ternary complexes of substrates with mutant AK, NMR studies or detailed X-ray crystallographic studies would be required, and circular dichroism and ORD studies must be regarding the structural change in our Lys-, Thr-, Val-, and Leu-mutants.  Our future efforts will be directed toward the other key residues required for the physiological function to define the interaction with substrates and essential amino acid residues.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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Figure Legends

 

Fig. 1. X-ray model

 

Fig. 2. NMR model

 

Fig. 3. Kim's model

 

Fig. 4. The artificial gene of human adenylate kinase

 

Fig. 5. Construction of pMEX8-hAK1 vector

 

Fig. 6. Random site-directed mutagenesis

            A flow chart of each experimental step.  Three small open circles in the         middle of the primer had a mismatched random XXY codon representing         a target residue, which was annealed to complement template DNA.  The             bold part of the outer line of homoduplex mutant pMEX8-hAK1 after            site-directed mutagenesis consisted of dCTPαS instead of dCTP.  The          accomplishment of synthesized homoduplex mutant strains was examined          by transformation on LB plates containing ampicillin.  After screening              of           mutants by DNA cycle sequence, the mutants were cultured, expressed            without recombination, and purified with chromatography as described in        the text.

 

Fig. 7. DNA sequence results of random mutations at Lys194 residue.

            DNA ladder patterns of WTAK and mutants for target codons are         respectively denoted.

 

Fig. 8. SDS-PAGE (The purified AK protein by SDS-PAGE electrophoresis)

 

Fig. 9. Kinetic analysis in the forward direction

 

 

 

 

 

 

Fig. 10.      The side chains of Lys9, Lys21, Lys27, Lys31, Lys63, Lys131, and        Lys194 are depicted in a proposed model of the binding sites for AMP        and MgATP in adenylate kinase by Kim et al. (1990).  These lysine             residues could take part in a phosphoryl transfer reaction between             MgATP2- and AMP2-, in cooperation with the other active site residues,       such as arginine residues (Kim et al., 1990).

 

Fig. 11.  Model of binding sites for AMP and MgATP in adenylate kinase proposed            by Kim et al.  Thr35 and Thr39 residues are denoted in the model.

 

Fig. 12.      A schematic depiction of the substrate binding sites for MgATP2- and         AMP2- on human adenylate kinase as deduced by Kim et al. (1990).  The           locations of Val182, Val186, Cys187, Leu190, and Leu193 residues are            depicted on the left side of this model.  The flanking C-terminal α          helices implicated in the catalytic mechanism in this study could                                                                                                             

            participate in a phosphoryl transfer reaction between MgATP2- and           AMP2-, in cooperation with other active site residues, such as arginine at         positions 44, 97, 132, and 138 (Kim et al., 1990) and lysine at positions 9,            21, 27, 31, 63, 131, and 194 (Ayabe et al., 1997). 

 

 

 

Table 1.  Sequence homology among the ATP-binding region of adenylate kinase             and segments of other proteins.

 

Table 2.  Summary of site-directed random mutagenesis

 

Table 3.  Summary of kinetic parameters of wild-type AK (WATK) and mutant AK

 

Table 4.  Summary of site-directed random mutagenesis

 

Table 5.  Summary of kinetic parameters of wild-type AK (WATK) and mutant AK

 

Table 6.  Summary of site-directed random mutagenesis

 

Table 7.  Summary of kinetic parameters of wild-type AK (WATK) and mutant AK