Mechanism of aminoglycoside antibiotic kinase APH(3')-IIIa: role of the nucleotide positioning loop

The aminoglycoside antibiotic resistance kinases (APHs) and the Ser/Thr/Tyr protein kinases share structural and functional homology but very little primary sequence conservation (<5%). A region of structural, but not amino acid sequence, homology is the nucleotide positioning loop (NPL) that closes down on the enzyme active site upon binding of ATP. This loop region has been implicated in facilitating phosphoryl transfer in protein kinases; however, there is no primary sequence conservation between APHs and protein kinases in the NPL. There is an invariant Ser residue in all APH NPL regions, however. This residue in APH(3')-IIIa (Ser27), an enzyme widespread in aminoglycoside-resistant Enterococci, Streptococci, and Staphylococci, directly interacts with the beta-phosphate of ATP through the Ser hydroxymethyl group and the amide hydrogen in the 3D structure of the enzyme. Mutagenesis of this residue to Ala and Pro supported a role for the Ser amide hydrogen in nucleotide capture and phosphoryl transfer. A molecular model of the proposed dissociative transition state, which is consistent with all of the available mechanistic data, suggested a role for the amide of the adjacent Met26 in phosphoryl transfer. Mutagenesis studies confirmed the importance of the amide hydrogen and suggest a mechanism where Ser27 anchors the ATP beta-phosphate facilitating bond breakage with the gamma-phosphate during formation of the metaphosphate-like transition, which is stabilized by interaction with the amide hydrogen of Met26. The APH NPL therefore acts as a lever, promoting phosphoryl transfer to the aminoglycoside substrate, with the biological outcome of clinically relevant antibiotic resistance.     

Establishing the principles of recognition in the adenine-binding region of an aminoglycoside antibiotic kinase [APH(3')-IIIa

The protein-based molecular recognition of the adenine ring has implications throughout biological systems. In this paper, we discuss the adenine-binding region of an aminoglycoside antibiotic kinase [APH(3')-IIIa], which serves as an excellent model system for proteins that bind the adenine ring. This enzyme employs a hydrogen-bonding network involving water molecules along with enzyme backbone/side-chain atoms and a pi-pi stacking interaction to recognize the adenine ring. Our approach utilized site-directed mutagenesis, adenosine analogues and a variety of biophysical methods to probe the contacts in the adenine-binding region of APH(3')-IIIa. The results point to the polar nature of an adenine-Met90 contact in this binding pocket and the important role that Met90, the "gatekeeper" residue in structurally similar Ser/Thr protein kinases, plays in adenine binding. The results also suggest that small changes in the structure of the adenine ring can lead to significant changes in the ability of these analogues to occupy the adenine-binding region of the enzyme. Additional computational experiments indicate that both size and electronic factors are important in the binding of aromatic systems in this interaction-rich pocket. The principles governing adenine recognition established in this study may be applied to other protein-ligand complexes and used to navigate future studies directed at discovering potent and selective inhibitors of APH-type enzymes.     

Molecular determinants of antibiotic recognition and resistance by aminoglycoside phosphotransferase (3')-IIIa: a calorimetric and mutational analysis

The growing threat from the emergence of multidrug resistant pathogens highlights a critical need to expand our currently available arsenal of broad-spectrum antibiotics. In this connection, new antibiotics must be developed that exhibit the abilities to circumvent known resistance pathways. An important step toward achieving this goal is to define the key molecular interactions that govern antibiotic resistance. Here, we use site-specific mutagenesis, coupled with calorimetric, NMR, and enzymological techniques, to define the key interactions that govern the binding of the aminoglycoside antibiotics neomycin and kanamycin B to APH(3')-IIIa (an antibiotic phosphorylating enzyme that confers resistance). Our mutational analyses identify the D261, E262, and C-terminal F264 residues of the enzyme as being critical for recognition of the two drugs as well as for the manifestation of the resistance phenotype. In addition, the E160 residue is more important for recognition of kanamycin B than neomycin, with mutation of this residue partially restoring sensitivity to kanamycin B but not to neomycin. By contrast, the D193 residue partially restores sensitivity to neomycin but not to kanamycin B, with the origins of this differential effect being due to the importance of D193 for catalyzing the phosphorylation of neomycin. These collective mutational results, coupled with (15)N NMR-derived pK(a) and calorimetrically derived binding-linked drug protonation data, identify the 1-, 3-, and 2'-amino groups of both neomycin and kanamycin B as being critical functionalities for binding to APH(3')-IIIa. These drug amino functionalities represent potential sites of modification in the design of next-generation compounds that can overcome APH(3')-IIIa-induced resistance.     

ATP binding enables broad antibiotic selectivity of aminoglycoside phosphotransferase(3')-IIIa: an elastic network analysis

The bacterial enzyme aminoglycoside phosphotransferase(3′)-IIIa (APH) confers resistance against a wide range of aminoglycoside antibiotics. In this study, we use the Gaussian network model to investigate how the binding of nucleotides and antibiotics influences the dynamics and thereby the ligand binding properties of APH. Interestingly, in NMR experiments, the dynamics differ significantly in various APH complexes, although crystallographic studies indicate that no larger conformational changes occur upon ligand binding. Isothermal titration calorimetry also shows different thermodynamic contributions to ligand binding. Formation of aminoglycoside-APH complexes is enthalpically driven, while the enthalpic change upon aminoglycoside binding to the nucleotide-APH complex is much smaller. The differential effects of nucleotide binding and antibiotic binding to APH can be explained theoretically by single-residue fluctuations and correlated motions of the enzyme. The surprising destabilization of β-sheet residues upon nucleotide binding, as seen in hydrogen/deuterium exchange experiments, shows that the number of closest neighbors does not fully explain residue flexibility. Additionally, we must consider correlated motions of dynamic protein domains, which show that not only connectivity but also the overall protein architecture is important for protein dynamics.     

6-hydroxy to 6'-amino tethered ring-to-ring macrocyclic aminoglycosides as probes for APH(3')-IIIa kinase

Based on molecular modeling and available X-ray structure data on aminoglycosides complexed with a bacterial ribosomal surrogate or with a kinase, two analogues of paromomycin were prepared by tethering the 6-OH and the 6'-NH(2) group with a five-carbon bridge. Only one of two possible hydroxyl groups was phosphorylated by the kinase. The application of ring closure metathesis is presented for the first time to construct bridged macrocyclic analogues in the aminoglycoside series.     

The COOH terminus of aminoglycoside phosphotransferase (3')-IIIa is critical for antibiotic recognition and resistance.

The aminoglycoside phosphotransferases (APHs) are widely distributed among pathogenic bacteria and are employed to covalently modify, and thereby detoxify, the clinically relevant aminoglycoside antibiotics. The crystal structure for one of these aminoglycoside kinases, APH(3')-IIIa, has been determined in complex with ADP and analysis of the electrostatic surface potential indicates that there is a large anionic depression present adjacent to the terminal phosphate group of the nucleotide. This region also includes a conserved COOH-terminal alpha-helix that contains the COOH-terminal residue Phe(264). We report here mutagenesis and computer modeling studies aimed at examining the mode of aminoglycoside binding to APH(3')-IIIa. Specifically, seven site mutants were studied, five from the COOH-terminal helix (Asp(261), Glu(262), and Phe(264)), and two additional residues that line the wall of the anionic depression (Tyr(55) and Arg(211)). Using a molecular modeling approach, six ternary complexes of APH(3')-IIIa.ATP with the antibiotics, kanamycin, amikacin, butirosin, and ribostamycin were independently constructed and these agree well with the mutagenesis data. The results obtained show that the COOH-terminal carboxylate of Phe(264) is critical for proper function of the enzyme. Furthermore, these studies demonstrate that there exists multiple binding modes for the aminoglycosides, which provides a molecular basis for the broad substrate- and regiospecificity observed for this enzyme.     

Mechanism of dihydroneopterin aldolase: functional roles of the conserved active site glutamate and lysine residues

Dihydroneopterin aldolase (DHNA) catalyzes the conversion of 7,8-dihydroneopterin (DHNP) to 6-hydroxymethyl-7,8-dihydropterin (HP) in the folate biosynthetic pathway. There are four conserved active site residues at the active site, E22, Y54, E74, and K100 in Staphylococcus aureus DHNA (SaDHNA), corresponding to E21, Y53, E73, and K98, respectively, in Escherichia coli DHNA (EcDHNA). The functional roles of the conserved glutamate and lysine residues have been investigated by site-directed mutagenesis in this work. E22 and E74 of SaDHNA and E21, E73, and K98 of EcDHNA were replaced with alanine. K100 of SaDHNA was replaced with alanine and glutamine. The mutant proteins were characterized by equilibrium binding, stopped-flow binding, and steady-state kinetic analyses. For SaDHNA, none of the mutations except E74A caused dramatic changes in the affinities of the enzyme for the substrate or product analogues or the rate constants. The Kd values for SaE74A were estimated to be >3000 microM, suggesting that the Kd values of the mutant are at least 100 times those of the wild-type enzyme. For EcDHNA, the E73A mutation increased the Kd values for the substrate or product analogues neopterin (MP), monapterin (NP), and 6-hydroxypterin (HPO) by factors of 340, 160, and 5600, respectively, relative to those of the wild-type enzyme. The K98A mutation increased the Kd values for NP, MP, and HPO by factors of 14, 3.6, and 230, respectively. The E21A mutation increased the Kd values for NP and HPO by factors of 2.2 and 42, respectively, but decreased the Kd value for MP by a factor of 3.3. The E22 (E21) and K100 (K98) mutations decreased the kcat values by factors of 1.3-2 x 10(4). The E74 (E73) mutation decreased in the kcat values by factors of approximately 10. The results suggested that E74 of SaDHNA and E73 of EcDHNA are important for substrate binding, but their roles in catalysis are minor. In contrast, E22 and K100 of SaDHNA are important for catalysis, but their roles in substrate binding are minor. On the other hand, E21 and K98 of EcDHNA are important for both substrate binding and catalysis.     

Detection of specific solvent rearrangement regions of an enzyme: NMR and ITC studies with aminoglycoside phosphotransferase(3')-IIIa

This work describes differential effects of solvent in complexes of the aminoglycoside phosphotransferase(3')-IIIa (APH) with different aminoglycosides and the detection of change in solvent structure at specific sites away from substrates. Binding of kanamycins to APH occurs with a larger negative DeltaH in H2O relative to D2O (DeltaDeltaH(H2O-D2O) < 0), while the reverse is true for neomycins. Unusually large negative DeltaCp values were observed for binding of aminoglycosides to APH. DeltaCp for the APH-neomycin complex was -1.6 kcal x mol(-1) x deg(-1). A break at 30 degrees C was observed in the APH-kanamycin complex yielding DeltaCp values of -0.7 kcal x mol(-1) x deg(-1) and -3.8 kcal x mol(-1) x deg(-1) below and above 30 degrees C, respectively. Neither the change in accessible surface area (DeltaASA) nor contributions from heats of ionization were sufficient to explain the large negative DeltaCp values. Most significantly, 15N-1H HSQC experiments showed that temperature-dependent shifts of the backbone amide protons of Leu 88, Ser 91, Cys 98, and Leu143 revealed a break at 30 degrees C only in the APH-kanamycin complex in spectra collected between 21 degrees C and 38 degrees C. These amino acids represent solvent reorganization sites that experience a change in solvent structure in their immediate environment as structurally different ligands bind to the enzyme. These residues were away from the substrate binding site and distributed in three hydrophobic patches in APH. Overall, our results show that a large number of factors affect DeltaCp and binding of structurally different ligand groups cause different solvent structure in the active site as well as differentially affecting specific sites away from the ligand binding site.     

Aminoglycoside 2'-phosphotransferase IIIa (APH(2')-IIIa) prefers GTP over ATP: structural templates for nucleotide recognition in the bacterial aminoglycoside-2' kinases

Contrary to the accepted dogma that ATP is the canonical phosphate donor in aminoglycoside kinases and protein kinases, it was recently demonstrated that all members of the bacterial aminoglycoside 2'-phosphotransferase IIIa (APH(2')) aminoglycoside kinase family are unique in their ability to utilize GTP as a cofactor for antibiotic modification. Here we describe the structural determinants for GTP recognition in these enzymes. The crystal structure of the GTP-dependent APH(2')-IIIa shows that although this enzyme has templates for both ATP and GTP binding superimposed on a single nucleotide specificity motif, access to the ATP-binding template is blocked by a bulky tyrosine residue. Substitution of this tyrosine by a smaller amino acid opens access to the ATP template. Similar GTP binding templates are conserved in other bacterial aminoglycoside kinases, whereas in the structurally related eukaryotic protein kinases this template is less conserved. The aminoglycoside kinases are important antibiotic resistance enzymes in bacteria, whose wide dissemination severely limits available therapeutic options, and the GTP binding templates could be exploited as new, previously unexplored targets for inhibitors of these clinically important enzymes.     

Probing the roles of active site residues in the 3'-5' exonuclease of the Werner syndrome protein

Werner syndrome is a premature aging disease caused by mutations in the WS gene and a deficiency in the function of Werner protein (WRN). The lack of WRN results in a cellular phenotype of genomic instability. WRN belongs to the RecQ DNA helicase family, but unlike other RecQ family members it possesses a functional exonuclease domain. We determined the crystal structure of mWRNexo (residues 31-238) bound to Zn(2+) and the sulfate ion. Compared with the structure of human WRNexo (hWRNexo), notable conformational changes were observed in several active site residues in an H5-H6 loop and in helices H6 and H7 of mWRNexo, presumably because of the presence of sulfate, which mimics the phosphate of substrate DNA. In particular, the side chains of Lys(185) and Tyr(206) were reoriented toward the Zn(2+) ion, whereas the side chain of Arg(190) pointed away from the active site center. Mutational analysis of these conserved residues abolished WRN exonuclease activity, suggesting that these residues play a critical role in the WRNexo activity. Based on substrate modeling and mutational analyses, we propose a mechanism by which WRNexo becomes activated upon substrate DNA binding. We also describe the low resolution trimeric structure of mouse WRNexoL (mWRNexoL, residues 31-330), as elucidated by small angle x-ray scattering (SAXS) analyses.     

Molecular mechanism of the enterococcal aminoglycoside 6'-N-acetyltransferase': role of GNAT-conserved residues in the chemistry of antibiotic inactivation

The Gram-positive pathogen Enterococcus faecium is intrinsically resistant to aminoglycoside antibiotics due to the presence of a chromosomally encoded aminoglycoside 6'-N-acetyltransferase [AAC(6')-Ii]. This enzyme is a member of the GCN5-related N-acetyltransferase (GNAT) superfamily and is therefore structurally homologous to proteins that catalyze acetyl transfer to diverse acyl-accepting substrates. This study reports the investigation of several potential catalytic residues that are present in the AAC(6')-Ii active site and also conserved in many GNAT enzymes. Site-directed mutagenesis of Glu72, His74, Leu76, and Tyr147 with characterization of the purified site mutants gave valuable information about the roles of these amino acids in acetyl transfer chemistry. More specifically, steady-state kinetic analysis of protein activity, solvent viscosity effects, pH studies, and antibiotic resistance profiles were all used to assess the roles of Glu72 and His74 as potential active site bases, Tyr147 as a general acid, and the importance of the amide NH group of Leu76 in transition-state stabilization. Taken together, our results indicate that Glu72 is not involved in general base catalysis, but is instead critical for the proper positioning and orientation of aminoglycoside substrates in the active site. Similarly, His74 is also not acting as the active site base, with pH studies revealing that this residue must be protonated for optimal AAC(6')-Ii activity. Mutation of Tyr147 was found not to affect the chemical step of catalysis, and our results were not consistent with this residue acting as a general acid. Last, the amide NH group of Leu76 is implicated in important interactions with acetyl-CoA and transition-state stabilization. In summary, the work described here provides important information regarding the molecular mechanism of AAC(6')-Ii catalysis that allows us to contrast our findings with those of other GNAT proteins and to demonstrate that these enzymes use a variety of chemical mechanisms to accelerate acyl transfer.     

The roles of conserved aromatic amino-acid residues in the active site of human lysozyme: a site-specific mutagenesis study

In order to probe the roles of Tyr-63, Trp-64 and Trp-109 in the active site of human lysozyme (peptidoglycan N-acetylmuramoylhydrolase, EC 3.2.1.17), six human lysozymes containing a mutation, Tyr-63 to Leu, Trp-64 to Phe or Tyr, Trp-109 to Phe or Tyr, and Glu-35 to Asp, were newly synthesized and their immunological and enzymatical activities were examined in comparison with the native enzyme. Enzymatic characterization indicated: (i) that the existences of an aromatic residue at position 63 and a tryptophan residue at position 64 are essential for the effective hydrolysis of glycol chitin substrate, but not for the lysis of bacterial substrate; (ii) that the conversion of Trp-109 to Phe or Tyr reduces the maximal velocity of the lytic reaction to 25% of the wild-type enzyme; however, the apparent affinity constant is not affected. Further, the difference between the activity against the charged substrate and that against the non-charged substrate was discussed from a viewpoint of the electrostatic interaction between enzyme and substrate.     

Molecular determinants of affinity for aminoglycoside binding to the aminoglycoside nucleotidyltransferase(2')-Ia

One of the most commonly occurring aminoglycoside resistance enzymes is aminoglycoside 2'-O-nucleotidyltransferase [ANT(2')]. In the present study molecular determinants of affinity and specificity for aminoglycoside binding to this enzyme are investigated using isothermal titration calorimetry (ITC). Binding of aminoglycosides is enthalpically driven accompanied by negative entropy changes. The presence of metal-nucleotide increases the affinity for all but one of the aminoglycosides studied but has no effect on specificity. The substituents at positions 1, 2', and 6' are important determinants of substrate specificity. An amino group at these positions leads to greater affinity. No correlation is observed between the change in affinity and enthalpy. At the 2' position greater affinity results from a more negative enthalpy for an aminoglycoside containing an amino rather than a hydroxyl at that position. At the 6' position the greater affinity for an aminoglycoside containing an amino substituent results from a less disfavorable entropic contribution. The thermodynamic basis for the change in affinity at position 1 could not be determined because of the weak binding of one of the aminoglycoside substrates, amikacin. The effect of increasing osmotic stress on affinity was used to determine that a net release of approximately four water molecules occurs when tobramycin binds to ANT(2'). No measurable net change in the number of bound water molecules is observed when neomycin binds the enzyme. Data acquired in this work provide the rationale for the ability of ANT(2') to confer resistance against kanamycins but not neomycins.     

Influence of the conserved active site residues of histidyl tRNA synthetase on the mechanism of aminoacylation reaction

The relation between the conservation of active site residues and the molecular mechanism of aminoacylation reaction is an unexplored problem. In the present paper, the influences of the conserved active site residues on the reaction mechanism as well as the electrostatic potential near the reaction center are analyzed for Histidyl tRNA synthetase from Escherichia coli, Thermus thermophilus and Staphylococcus aureus. While the primary structures show both convergence as well as divergence, the secondary level structures of the active sites of the three species show considerable conservation in the respective structural organizations. The conserved active site residues near the reaction center, which have a major role in the reaction mechanism and catalysis, retain their specific position and orientation relative to the substrate in the three species. In order to understand the influence of different conserved and nonconserved residues near the reaction center, two different models are considered. First, a large model of active site with the substrates, Mg²⁺ ions and water is constructed in which the first shell residues (including both conserved as well as nonconserved) near the reaction center are studied. From the large model, a smaller model is constructed for reaction path modeling individually for three species. Validation of the smaller model is carried out by comparing the energy surfaces of large and small models as a function of reaction coordinates. Further, the electrostatic potential near the reaction center for the large and small model are compared. The transition state structures of the activation step of aminoacylation reaction for E. coli, T. thermophilus and S. aureus are calculated using the combined ab-initio/semi-empirical calculation. The similarity of the energy profiles as a function of the relevant reaction coordinate and the orientation of the catalytic residue, Arg259, indicate that the reaction mechanisms are identical which are guided by the strikingly similar structural pattern formed by conserved residues for three species. The energy surfaces have close resemblance in three species and present a clear perspective that how the reaction proceeds with the aid of different conserved residues. The study of electrostatic potential confirms this view. The present study provides an understanding of the relationship between the conservation of residues and the efficient reaction mechanism of aminoacylation reaction.     

Differences in the roles of conserved glutamic acid residues in the active site of human class 3 and class 2 aldehyde dehydrogenases.

Although the three-dimensional structure of the dimeric class 3 rat aldehyde dehydrogenase has recently been published (Liu ZJ et al., 1997, Nature Struct Biol 4:317-326), few mechanistic studies have been conducted on this isoenzyme. We have characterized the enzymatic properties of recombinant class 3 human stomach aldehyde dehydrogenase, which is very similar in amino acid sequence to the class 3 rat aldehyde dehydrogenase. We have determined that the rate-limiting step for the human class 3 isozyme is hydride transfer rather than deacylation as observed for the human liver class 2 mitochondrial enzyme. No enhancement of NADH fluorescence was observed upon binding to the class 3 enzyme, while fluorescence enhancement of NADH has been previously observed upon binding to the class 2 isoenzyme. It was also observed that binding of the NAD cofactor inhibited the esterase activity of the class 3 enzyme while activating the esterase activity of the class 2 enzyme. Site-directed mutagenesis of two conserved glutamic acid residues (209 and 333) to glutamine residues indicated that, unlike in the class 2 enzyme, Glu333 served as the general base in the catalytic reaction and E209Q had only marginal effects on enzyme activity, thus confirming the proposed mechanism (Hempel J et al., 1999, Adv Exp Med Biol 436:53-59). Together, these data suggest that even though the subunit structures and active site residues of the isozymes are similar, the enzymes have very distinct properties besides their oligomeric state (dimer vs. tetramer) and substrate specificity.     

A conserved 3'----5' exonuclease active site in prokaryotic and eukaryotic DNA polymerases

The 3'----5' exonuclease active site of E. coli DNA polymerase I is predicted to be conserved for both prokaryotic and eukaryotic DNA polymerases based on amino acid sequence homology. Three amino acid regions containing the critical residues in the E. coli DNA polymerase I involved in metal binding, single-stranded DNA binding, and catalysis of the exonuclease reaction are located in the amino-terminal half and in the same linear arrangement in several prokaryotic and eukaryotic DNA polymerases. Site-directed mutagenesis at the predicted exonuclease active site of the phi 29 DNA polymerase, a model enzyme for prokaryotic and eukaryotic alpha-like DNA polymerases, specifically inactivated the 3'----5' exonuclease activity of the enzyme. These results reflect a high evolutionary conservation of this catalytic domain. Based on structural and functional data, a modular organization of enzymatic activities in prokaryotic and eukaryotic DNA polymerases is also proposed.     

Yeast pyruvate kinase: essential lysine residues in the active site

1. Yeast pyruvate kinase was purified to near homogeneity and subjected to chemical modification by trinitrobenzenesulfonate and by P1, P2-bis (5' pyridoxal) diphosphate. 2. Labeled peptides were isolated and their amino acid composition was determined. 3. The results suggest that yeast pyruvate kinase has an essential lysine residue, and that this residue is in a location equivalent to an essential lysine described in the muscle enzyme. 4. Protection experiments indicate that this lysine is located at the nucleotide binding site.     

Coenzyme A binding to the aminoglycoside acetyltransferase (3)-IIIb increases conformational sampling of antibiotic binding site

NMR spectroscopy experiments and molecular dynamics simulations were performed to describe the dynamic properties of the aminoglycoside acetyltransferase (3)-IIIb (AAC) in its apo and coenzyme A (CoASH) bound forms. The (15)N-(1)H HSQC spectra indicate a partial structural change and coupling of the CoASH binding site with another region in the protein upon the CoASH titration into the apo enzyme. Molecular dynamics simulations indicate a significant structural and dynamic variation of the long loop in the antibiotic binding domain in the form of a relatively slow (250 ns), concerted opening motion in the CoASH-enzyme complex and that binding of the CoASH increases the structural flexibility of the loop, leading to an interchange between several similar equally populated conformations.     

Mutagenesis of two acidic active site residues in human muscle creatine kinase: implications for the catalytic mechanism

Creatine kinase (CK) catalyzes the reversible phosphorylation of the guanidine substrate, creatine, by MgATP. Although several X-ray crystal structures of various isoforms of creatine kinase have been published, the detailed catalytic mechanism remains unresolved. A crystal structure of the CK homologue, arginine kinase (AK), complexed with the transition-state analogue (arginine-nitrate-ADP), has revealed two carboxylate amino acid residues (Glu225 and Glu314) within 2.8 A of the proposed transphosphorylation site. These two residues are the putative catalytic groups that may promote nucleophilic attack by the guanidine amino group on the gamma-phosphate of ATP. From primary sequence alignments of arginine kinases and creatine kinases, we have identified two homologous creatine kinase acidic amino acid residues (Glu232 and Asp326), and these were targeted for examination of their potential roles in the CK mechanism. Using site-directed mutagenesis, we have made several substitutions at these two positions. The results indicate that of these two residues the Glu232 is the likely catalytic residue while Asp326 likely performs a role in properly aligning substrates for catalysis.     

The tryptophane residues of dimeric arginine kinase: roles of Trp-208 and Trp-218 in active site and conformation stability

Roles of the two tryptophane residues of dimeric arginine kinase (AK) were individually investigated by site-directed mutagenesis. Both residues were fully conserved in the phosphogen kinase family and the mutant proteins were analyzed by enzyme kinetics, fluorescence spectroscopy, fluorescence quenching experiments, thermal stability and conformational stability. Our studies revealed that Trp-218 was located at the active site of AK and was the major fluorescence contributor (96.9%). Single replacement of this residue by alanine led to almost complete inactivation of the enzyme. In addition, a decrease in the melting temperature in differential scanning calorimetry (DSC) profiles and the equilibrium studies in guanidine hydrochloride (GdnHCl) denaturation after mutagenesis also suggested that Trp-218 takes part in stabilizing the conformational structure of AK. Although another tryptophane, Trp-208 was not located at the active sites, it may take part in maintaining the correct dimer conformation for catalysis. Replacement of this tryptophane by alanine decreased the activity to 70.3% and made it susceptible to heat and denaturants, such as GdnHCl. In addition, Trp-208 also seemed to play an important role in correct protein folding.