Mechanistic and structural analyses of the roles of Arg409 and Asp402 in the reaction of the flavoprotein nitroalkane oxidase

The flavoprotein nitroalkane oxidase (NAO) catalyzes the oxidation of primary and secondary nitroalkanes to the corresponding aldehydes and ketones. The enzyme is a homologue of acyl-CoA dehydrogenase. Asp402 in NAO has been proposed to be the active site base responsible for removing the substrate proton in the first catalytic step; structurally it corresponds to the glutamate which acts as the base in medium chain acyl-CoA dehydrogenase. In the active site of NAO, the carboxylate of Asp402 forms an ionic interaction with the side chain of Arg409. The R409K enzyme has now been characterized kinetically and structurally. The mutation results in a decrease in the rate constant for proton abstraction of 100-fold. Analysis of the three-dimensional structure of the R409K enzyme, determined by X-ray crystallography to a resolution of 2.65 A, shows that the critical structural change is an increase in the distance between the carboxylate of Asp402 and the positively charged nitrogen in the side chain of the residue at position 409. The D402E mutation results in a smaller decrease in the rate constant for proton abstraction of 18-fold. The structure of the D402E enzyme, determined at 2.4 A resolution, shows that there is a smaller increase in the distance between Arg409 and the carboxylate at position 402, and the interaction of this residue with Ser276 is perturbed. These results establish the critical importance of the interaction between Asp402 and Arg409 for proton abstraction by nitroalkane oxidase.     

Functional role of residues in the helix B' region of cytochrome P450 2B1

While several flavoproteins will oxidize nitroalkanes in addition to their physiological substrates, nitroalkane oxidase (NAO) is the only one which does not require the anionic nitroalkane. This, in addition to the induction of NAO by nitroethane seen in Fusarium oxysporum, suggests that oxidation of a nitroaliphatic species is the physiological role of the enzyme. Mechanistic studies of the reaction with nitroethane as substrate have established many of the details of the enzymatic reaction. The enzyme is unique in being the only flavoprotein to date for which a carbanion is definitively established as an intermediate in catalysis. Recent structural analyses show that NAO is homologous to the acyl-CoA dehydrogenase and acyl-CoA oxidase families of enzymes. In NAO, the glutamate which acts as the active site base in the latter enzymes is replaced by an aspartate.     

Kinetic and crystallographic analysis of the key active site acid/base arginine in a soluble fumarate reductase

There is now overwhelming evidence supporting a common mechanism for fumarate reduction in the respiratory fumarate reductases. The X-ray structures of substrate-bound forms of these enzymes indicate that the substrate is well positioned to accept a hydride from FAD and a proton from an arginine side chain. Recent work on the enzyme from Shewanella frigidimarina [Doherty, M. K., Pealing, S. L., Miles, C. S., Moysey, R., Taylor, P., Walkinshaw, M. D., Reid, G. A., and Chapman, S. K. (2000) Biochemistry 39, 10695-10701] has strengthened the assignment of an arginine (Arg402) as the proton donor in fumarate reduction. Here we describe the crystallographic and kinetic analyses of the R402A, R402K, and R402Y mutant forms of the Shewanella enzyme. The crystal structure of the R402A mutant (2.0 A resolution) shows it to be virtually identical to the wild-type enzyme, apart from the fact that a water molecule occupies the position previously taken by part of the guanidine group of R402. Although structurally similar to the wild-type enzyme, the R402A mutant is inactive under all the conditions that were studied. This implies that a water molecule, in this position in the active site, cannot function as the proton donor for fumarate reduction. In contrast to the R402A mutation, both the R402K and R402Y mutant enzymes are active. Although this activity was at a very low level (at pH 7.2 some 10(4)-fold lower than that for the wild type), it does imply that both lysine and tyrosine can fulfill the role of an active site proton donor, albeit very poorly. The crystal structures of the R402K and R402Y mutant enzymes (2.0 A resolution) show that distances from the lysine and tyrosine side chains to the nearest carbon atom of fumarate are approximately 3.5 A, clearly permitting proton transfer. The combined results from mutagenesis, crystallographic, and kinetic studies provide formidable evidence that R402 acts as both a Lewis acid (stabilizing the build-up of negative charge upon hydride transfer from FAD to fumarate) and a Br?nsted acid (donating the proton to the substrate to complete the formation of succinate).     

Characterization of active site residues of nitroalkane oxidase

The flavoenzyme nitroalkane oxidase catalyzes the oxidation of primary and secondary nitroalkanes to the corresponding aldehydes and ketones plus nitrite. The structure of the enzyme shows that Ser171 forms a hydrogen bond to the flavin N5, suggesting that it plays a role in catalysis. Cys397 and Tyr398 were previously identified by chemical modification as potential active site residues. To more directly probe the roles of these residues, the S171A, S171V, S171T, C397S, and Y398F enzymes have been characterized with nitroethane as substrate. The C397S and Y398 enzymes were less stable than the wild-type enzyme, and the C397S enzyme routinely contained a substoichiometric amount of FAD. Analysis of the steady-state kinetic parameters for the mutant enzymes, including deuterium isotope effects, establishes that all of the mutations result in decreases in the rate constants for removal of the substrate proton by ∼5-fold and decreases in the rate constant for product release of ∼2-fold. Only the S171V and S171T mutations alter the rate constant for flavin oxidation. These results establish that these residues are not involved in catalysis, but rather are required for maintaining the protein structure.     

Oxygen and hydrogen isotope effects in an active site tyrosine to phenylalanine mutant of peptidylglycine alpha-hydroxylating monooxygenase: mechanistic implications

Peptidylglycine alpha-hydroxylating monooxygenase (PHM) and dopamine beta-monooxygenase (DbetaM) are homologous copper-containing enzymes that catalyze an oxygen-dependent hydroxylation of peptide-extended glycine residues and phenethylamines, respectively. The mechanism whereby these enzymes activate molecular oxygen and the C-H bond of substrate has been the subject of numerous studies, and various mechanisms have been put forth. From the magnitude of (18)O isotope effects as a function of substrate structure in DbetaM, an active site tyrosine had been proposed to function in the reductive activation of Cu(II)-OOH to generate a reactive copper-oxo species [Tian et al. (1994) Biochemistry 33, 226]. The presence of a tyrosine residue, Y318, in the active site of PHM was subsequently confirmed from crystallographic studies [Prigge et al. (1997) Science 278, 1300]. We now report extensive kinetic and isotope effect studies on the Y318F mutant form of PHM, analyzing the role of this tyrosine in the catalytic mechanism. It is found that the Y318F mutant has intrinsic hydrogen and (18)O isotope effects that are within experimental error of the wild-type enzyme and that the mutation causes only a slight reduction in the rate constant for C-H bond cleavage. These findings, together with the recent demonstration that C-H activation in PHM is dominated by quantum mechanical tunneling [Francisco et al. (2002) J. Am. Chem. Soc. 124, 8194], necessitate a reexamination of plausible mechanisms for this unique class of copper enzymes.     

Kinetic studies of the mechanism of carbon-hydrogen bond breakage by the heterotetrameric sarcosine oxidase of Arthrobacter sp. 1-IN

The reaction of heterotetrameric sarcosine oxidase (TSOX) of Arthrobactor sp. 1-IN has been studied by stopped-flow spectroscopy, with particular emphasis on the reduction of the enzyme by sarcosine. Expression of the cloned gene encoding TSOX in Escherichia coli enables the production of TSOX on a scale suitable for stopped-flow studies. Treatment of the enzyme with sulfite provides the means for selective formation of a flavin-sulfite adduct with the covalent 8alpha-(N(3)-histidyl)-FMN. Formation of the sulfite-flavin adduct suppresses internal electron transfer between the noncovalent FAD (site of sarcosine oxidation) and the covalent FMN (site of enzyme oxidation) and thus enables detailed characterization of the kinetics of FAD reduction by sarcosine using stopped-flow methods. The rate of FAD reduction displays a simple hyperbolic dependence on sarcosine concentration. Studies in the pH range 6.5-10 indicate there are no kinetically influential ionizations in the enzyme-substrate complex. A plot of the limiting rate of flavin reduction/the enzyme-substrate dissociation constant (k(lim)/K(d)) versus pH is bell-shaped and characterized by two macroscopic pK(a) values of 7.4 +/- 0.1 and 10.4 +/- 0.2: potential candidates for the two ionizable groups are discussed with reference to the structure of monomeric sarcosine oxidase (MSOX). The kinetic data are discussed with reference to potential mechanisms for the oxidation of amine molecules by flavoenzymes. Additionally, kinetic isotope effect studies of the rate of C-H bond breakage suggest that a ground-state quantum tunneling mechanism for H-transfer, facilitated by the low-frequency thermal motions of the protein molecule, accounts for C-H bond cleavage by TSOX. TSOX thus provides another example of C-H bond breakage by ground-state quantum tunneling, driven by protein dynamics [vibrationally enhanced ground-state quantum tunneling (VEGST)], for the oxidation of amines by enzymes.     

Engineering water to act as an active site acid catalyst in a soluble fumarate reductase

The ability of an arginine residue to function as the active site acid catalyst in the fumarate reductase family of enzymes is now well-established. Recently, a dual role for the arginine during fumarate reduction has been proposed [Mowat, C. G., Moysey, R., Miles, C. S., Leys, D., Doherty, M. K., Taylor, P., Walkinshaw, M. D., Reid, G. A., and Chapman, S. K. (2001) Biochemistry 40, 12292-12298] in which it acts both as a Lewis acid in transition-state stabilization and as a Br?nsted acid in proton delivery. This proposal has led to the prediction that, if appropriately positioned, a water molecule would be capable of functioning as the active site Br?nsted acid. In this paper, we describe the construction and kinetic and crystallographic analysis of the Q363F single mutant and Q363F/R402A double mutant forms of flavocytochrome c(3), the soluble fumarate reductase from Shewanella frigidimarina. Although replacement of the active site acid, Arg402, with alanine has been shown to eliminate fumarate reductase activity, this phenomenon is partially reversed by the additional substitution of Gln363 with phenylalanine. This Gln --> Phe substitution in the inactive R402A mutant enzyme was designed to "push" a water molecule close enough to the substrate C3 atom to allow it to act as a Br?nsted acid. The 2.0 A resolution crystal structure of the Q363F/R402A mutant enzyme does indeed reveal the introduction of a water molecule at the correct position in the active site to allow it to act as the catalytic proton donor. The 1.8 A resolution crystal structure of the Q363F mutant enzyme shows a water molecule similarly positioned, which can account for its measured fumarate reductase activity. However, in this mutant enzyme Michaelis complex formation is impaired due to significant and unpredicted structural changes at the active site.     

Mechanistic and structural studies of H373Q flavocytochrome b2: effects of mutating the active site base

His373 in flavocytochrome b2 has been proposed to act as an active site base during the oxidation of lactate to pyruvate, most likely by removing the lactate hydroxyl proton. The effects of mutating this residue to glutamine have been determined to provide further insight into its role. The kcat and kcat/Klactate values for the mutant protein are 3 to 4 orders of magnitude smaller than the wild-type values, consistent with a critical role for His373. Similar effects are seen when the mutation is incorporated into the isolated flavin domain of the enzyme, narrowing the effects to lactate oxidation rather than subsequent electron transfers. The decrease of 3500-fold in the rate constant for reduction of the enzyme-bound FMN by lactate confirms this part of the reaction as that most effected by the mutation. The primary deuterium and solvent kinetic isotope effects for the mutant enzyme are significantly smaller than the wild-type values, establishing that bond cleavage steps are less rate-limiting in H373Q flavocytochrome b2 than in the wild-type enzyme. The structure of the mutant enzyme with pyruvate bound, determined at 2.8 A, provides a rationale for these effects. The orientation of pyruvate in the active site is altered from that seen in the wild-type enzyme. In addition, the active site residues Arg289, Asp 292, and Leu 286 have altered positions in the mutant protein. The combination of an altered active site and the small kinetic isotope effects is consistent with the slowest step in turnover being a conformational change involving a conformation in which lactate is bound unproductively.     

Structure-function studies of substrate oxidation by bovine serum amine oxidase: relationship to cofactor structure and mechanism.

The chemical mechanism of substrate oxidation, catalyzed by bovine serum amine oxidase, has been explored by a detailed investigation of structure-reactivity correlations. Past mechanistic studies, involving the reductive trapping of substrate to cofactor [Hartmann, C., & Klinman, J. P. (1987) J. Biol. Chem. 262, 962], implied the intermediacy of a substrate imine complex in the catalytic redox mechanism. These studies led to the proposal of a transamination mechanism for substrate oxidation, analogous to pyridoxal phosphate dependent enzymes. In pyridoxal phosphate catalyzed reactions, the transamination process involves the transient formation of a resonance-stabilized carbanion intermediate. Although evidence has been presented describing the participation of an active site base in bovine serum amine oxidase catalysis [Farnum, M. F., Palcic, M. M., & Klinman, J. P. (1986) Biochemistry 25, 1898], the nature of the intermediate derived from C-H bond cleavage has not been directly addressed. To examine this question, a structure-reactivity study was performed using a series of para-substituted benzylamines. Having prior knowledge of the intrinsic isotope effect for an enzymatic reaction permits calculation of microscopic rate constants from steady-state data [Palcic, M. M., & Klinman, J. P. (1983) Biochemistry 22, 5957]. Deuterium isotope effects on kcat and kcat/Km parameters were determined for all substrates, allowing for the calculation of rate constants for C-H bond cleavage (k3) and substrate dissociation constants (Kd). Pre-steady-state constants obtained for p-acetylbenzylamine, p-(trifluoromethyl)benzylamine, and unsubstituted benzylamine exhibited excellent agreement with values calculated from steady-state isotope effects.(ABSTRACT TRUNCATED AT 250 WORDS)     

Detection of large pKa perturbations of an inhibitor and a catalytic group at an enzyme active site, a mechanistic basis for catalytic power of many enzymes

Delta(5)-3-Ketosteroid isomerase catalyzes cleavage and formation of a C-H bond at a diffusion-controlled limit. By determining the crystal structures of the enzyme in complex with each of three different inhibitors and by nuclear magnetic resonance (NMR) spectroscopic investigation, we evidenced the ionization of a hydroxyl group (pK(a) approximately 16.5) of an inhibitor, which forms a low barrier hydrogen bond (LBHB) with a catalytic residue Tyr(14) (pK(a) approximately 11.5), and the protonation of the catalytic residue Asp(38) with pK(a) of approximately 4.5 at pH 6.7 in the interaction with a carboxylate group of an inhibitor. The perturbation of the pK(a) values in both cases arises from the formation of favorable interactions between inhibitors and catalytic residues. The results indicate that the pK(a) difference between catalytic residue and substrate can be significantly reduced in the active site environment as a result of the formation of energetically favorable interactions during the course of enzyme reactions. The reduction in the pK(a) difference should facilitate the abstraction of a proton and thereby eliminate a large fraction of activation energy in general acid/base enzyme reactions. The pK(a) perturbation provides a mechanistic ground for the fast reactivity of many enzymes and for the understanding of how some enzymes are able to extract a proton from a C-H group with a pK(a) value as high as approximately 30.     

Kinetic isotope effect studies on milk xanthine oxidase and on chicken liver xanthine dehydrogenase

The effect of isotopic substitution of the 8-H of xanthine (with 2H and 3H) on the rate of oxidation by bovine xanthine oxidase and by chicken xanthine dehydrogenase has been measured. V/K isotope effects were determined from competition experiments. No difference in H/T(V/K) values was observed between xanthine oxidase (3.59 +/- 0.1) and xanthine dehydrogenase (3.60 +/- 0.09). Xanthine dehydrogenase exhibited a larger T/D(V/K) value (0.616 +/- 0.028) than that observed for xanthine oxidase (0.551 +/- 0.016). Observed H/T(V/K) values for either enzyme are less than those H/T(V/K) values calculated with D/T(V/K) data. These discrepancies are suggested to arise from the presence of a rate-limiting step(s) prior to the irreversible C-H bond cleavage step in the mechanistic pathways of both enzymes. These kinetic complexities preclude examination of whether tunneling contributes to the reaction coordinate for the H-transfer step in each enzyme. No observable exchange of tritium with solvent is observed during the anaerobic incubation of [8-3H]xanthine with either enzyme, which suggests the reverse commitment to catalysis (Cr) is essentially zero. With the assumption of adherence to reduced mass relationships, the intrinsic deuterium isotope effect (Dk) for xanthine oxidation is calculated to be 7.4 +/- 0.7 for xanthine oxidase and 4.2 +/- 0.2 for xanthine dehydrogenase. By use of these values and steady-state kinetic data, the minimal rate for the hydrogen-transfer step is calculated to be approximately 75-fold faster than kcat for xanthine oxidase and approximately 10-fold faster than kcat for xanthine dehydrogenase. This calculated rate is consistent with data obtained by rapid-quench experiments with XO. A stoichiometry of 1.0 +/- 0.3 mol of uric acid/mol of functional enzyme is formed within the mixing time of the instrument (5-10 ms). The kinetic isotope effect data also permitted the calculation of the Kd values [Klinman, J. P., & Mathews, R. G. (1985) J. Am. Chem. Soc. 107, 1058-1060] for substrate dissociation, including all reversible steps prior to C-H bond cleavage. Values calculated for each enzyme (Kd = 120 microM) were found to be identical within experimental uncertainty.     

Subsite mapping of the human pancreatic alpha-amylase active site through structural, kinetic, and mutagenesis techniques

We report a multifaceted study of the active site region of human pancreatic alpha-amylase. Through a series of novel kinetic analyses using malto-oligosaccharides and malto-oligosaccharyl fluorides, an overall cleavage action pattern for this enzyme has been developed. The preferred binding/cleavage mode occurs when a maltose residue serves as the leaving group (aglycone sites +1 and +2) and there are three sugars in the glycon (-1, -2, -3) sites. Overall it appears that five binding subsites span the active site, although an additional glycon subsite appears to be a significant factor in the binding of longer substrates. Kinetic parameters for the cleavage of substrates modified at the 2 and 4' ' positions also highlight the importance of these hydroxyl groups for catalysis and identify the rate-determining step. Further kinetic and structural studies pinpoint Asp197 as being the likely nucleophile in catalysis, with substitution of this residue leading to an approximately 10(6)-fold drop in catalytic activity. Structural studies show that the original pseudo-tetrasaccharide structure of acarbose is modified upon binding, presumably through a series of hydrolysis and transglycosylation reactions. The end result is a pseudo-pentasaccharide moiety that spans the active site region with its N-linked "glycosidic" bond positioned at the normal site of cleavage. Interestingly, the side chains of Glu233 and Asp300, along with a water molecule, are aligned about the inhibitor N-linked glycosidic bond in a manner suggesting that these might act individually or collectively in the role of acid/base catalyst in the reaction mechanism. Indeed, kinetic analyses show that substitution of the side chains of either Glu233 or Asp300 leads to as much as a approximately 10(3)-fold decrease in catalytic activity. Structural analyses of the Asp300Asn variant of human pancreatic alpha-amylase and its complex with acarbose clearly demonstrate the importance of Asp300 to the mode of inhibitor binding.     

Identification of catalytic and substrate-binding site residues in Bacillus cereus ATCC7064 oligo-1,6-glucosidase.

Three active site residues (Asp199, Glu255, Asp329) and two substrate-binding site residues (His103, His328) of oligo-1,6-glucosidase (EC 3.2.1.10) from Bacillus cereus ATCC7064 were identified by site-directed mutagenesis. These residues were deduced from the X-ray crystallographic analysis and the comparison of the primary structure of the oligo-1,6-glucosidase with those of Saccharomyces carlsbergensis alpha-glucosidase, Aspergillus oryzae alpha-amylase and pig pancreatic alpha-amylase which act on alpha-1,4-glucosidic linkages. The distances between these putative residues of B. cereus oligo-1,6-glucosidase calculated from the X-ray analysis data closely resemble those of A. oryzae alpha-amylase and pig pancreatic alpha-amylase. A single mutation of Asp199-->Asn, Glu255-->Gln, or Asp329-->Asn resulted in drastic reduction in activity, confirming that three residues are crucial for the reaction process of alpha-1,6-glucosidic bond cleavage. Thus, it is identified that the basic mechanism of oligo-1,6-glucosidase for the hydrolysis of alpha-1,6-glucosidic linkage is essentially the same as those of other amylolytic enzymes belonging to Family 13 (alpha-amylase family). On the other hand, mutations of histidine residues His103 and His328 resulted in pronounced dissimilarity in catalytic function. The mutation His328-->Asn caused the essential loss in activity, while the mutation His103-->Asn yielded a mutant enzyme that retained 59% of the k0/Km of that for the wild-type enzyme. Since mutants of other alpha-amylases acting on alpha-1,4-glucosidic bond linkage lost most of their activity by the site-directed mutagenesis at their equivalent residues to His103 and His328, the retaining of activity by His103-->Asn mutation in B. cereus oligo-1,6-glucosidase revealed the distinguished role of His103 for the hydrolysis of alpha-1,6-glucosidic bond linkage.     

Characterization of two independent amino acid substitutions that disrupt the DNA repair functions of the yeast Apn1

The members of the Endo IV family of DNA repair enzymes, including Saccharomyces cerevisiae Apn1 and Escherichia coli endonuclease IV, possess the capacity to cleave abasic sites and to remove 3'-blocking groups at single-strand breaks via apurinic/apyrimidinic (AP) endonuclease and 3'-diesterase activities, respectively. In addition, Endo IV family members are able to recognize and incise oxidative base damages on the 5'-side of such lesions. We previously identified eight amino acid substitutions that prevent E. coli endonuclease IV from repairing damaged DNA in vivo. Two of these substitutions were glycine replacements of Glu145 and Asp179. Both Glu145 and Asp179 are among nine amino acid residues within the active site pocket of endonuclease IV that coordinate the position of a trinuclear Zn cluster required for efficient phosphodiester bond cleavage. We now report the first structure-function analysis of the eukaryotic counterpart of endonuclease IV, yeast Apn1. We show that glycine substitutions at the corresponding conserved amino acid residues of yeast Apn1, i.e., Glu158 and Asp192, abolish the biological function of this enzyme. However, these Apn1 variants do not exhibit the same characteristics as the corresponding E. coli mutants. Indeed, the Apn1 Glu158Gly mutant, but not the E. coli endonuclease IV Glu145Gly mutant, is able to bind DNA. Moreover, Apn1 Asp192Gly completely lacks enzymatic activity, while the activity of the E. coli counterpart Asp179Gly is reduced by approximately 40-fold. The data suggest that although yeast Apn1 and E. coli endonuclease IV exhibit a high degree of structural and functional similarity, differences exist within the active site pockets of these two enzymes.     

Structures of purine 2'-deoxyribosyltransferase, substrate complexes, and the ribosylated enzyme intermediate at 2.0 A resolution

The structure of class I N-deoxyribosyltransferase from Lactobacillus helveticus was determined by X-ray crystallography. Unlike class II N-deoxyribosyltransferases, which accept either purine or pyrimidine deoxynucleosides, class I enzymes are specific for purines as both the donor and acceptor base. Both class I and class II enzymes are highly specific for deoxynucleosides. The class I structure reveals similarities with the previously determined class II enzyme from Lactobacillus leichmanni [Armstrong, S. A., Cook, W. J., Short, S. A., and Ealick, S. E. (1996) Structure 4, 97-107]. The specificity of the class I enzyme for purine deoxynucleosides can be traced to a loop (residues 48-62), which shields the active site in the class II enzyme. In the class I enzyme, the purine base itself shields the active site from the solvent, while the smaller pyrimidine base cannot. The structure of the enzyme with a bound ribonucleoside shows that the nucleophilic oxygen atom of Glu101 hydrogen bonds to the O2' atom, rendering it unreactive and thus explaining the specificity for 2'-deoxynucleosides. The structure of a ribosylated enzyme intermediate reveals movements that occur during cleavage of the N-glycosidic bond. The structures of complexes with substrates and substrate analogues show that the purine base can bind in several different orientations, thus explaining the ability of the enzyme to catalyze alternate deoxyribosylation at the N3 or N7 position.     

Catalytic mechanism of Escherichia coli glycinamide ribonucleotide transformylase probed by site-directed mutagenesis and pH-dependent studies.

Site-directed mutagenesis followed by studies of the pH dependence of the kinetic parameters of the mutants has been used to probe the role of the active site residues and loops in catalysis by glycinamide ribonucleotide transformylase (EC 2.1.2.2). The analysis of the mutants of the strictly conserved active site residues, His108 and Asp144, revealed that His108 acts in a salt bridge with Asp144 as a general acid catalyst with a pK(a) value of 9.7. Asp144 also plays a key role in the preparation of the active site geometry for catalysis. The rate-limiting step in the pH range of 6-10 appears to be the catalytic steps involving tetrahedral intermediates, supported by the observation of a pL (L being H or D)-independent solvent deuterium isotope effect of 2. The ionization of the amino group of glycinamide ribonucleotide both as a free and as a bound form dominates the kinetic behavior at low pH. The analysis of a mutation, H121Q, within the loop spanning amino acids 111-131 suggests the closure of the loop is involved in the binding of the substrate. The kinetic behavior parallels pH effects revealed by a series of X-ray crystallographic structures of the apoenzyme and inhibitor-bound enzyme [Su, Y., Yamashita, M. M., Greasley, S. E. , Mullen, C. A., Shim, J. H., Jennings, P. A., Benkovic, S. J., and Wilson, I. A. (1998) J. Mol. Biol. 281, 485-499], permitting a more exact formulation of the probable catalytic mechanism.     

Identification of Catalytic and Substrate-binding Site Residues in Bacillus cereus ATCC7064 Oligo-1,6-glucosidase

Three active site residues (Asp199, Glu255, Asp329) and two substrate-binding site residues (His103, His328) of oligo-1,6-glucosidase (EC 3.2.1.10) from Bacillus cereus ATCC7064 were identified by site-directed mutagenesis. These residues were deduced from the X-ray crystallographic analysis and the comparison of the primary structure of the oligo-1,6-glucosidase with those of Saccharomyces carlsbergensis α-glucosidase, Aspergillus oryzae α-amylase and pig pancreatic α-amylase which act on α-1,4-glucosidic linkages. The distances between these putative residues of B. cereus oligo-1,6-glucosidase calculated from the X-ray analysis data closely resemble those of A. oryzae α-amylase and pig pancreatic α-amylase. A single mutation of Asp199→Asn, Glu255→Gln, or Asp329→Asn resulted in drastic reduction in activity, confirming that three residues are crucial for the reaction process of α-1,6-glucosidic bond cleavage. Thus, it is identified that the basic mechanism of oligo-1,6-glucosidase for the hydrolysis of α-1,6-glucosidic linkage is essentially the same as those of other amylolytic enzymes belonging to Family 13 (α-amylase family). On the other hand, mutations of histidine residues His103 and His328 resulted in pronounced dissimilarity in catalytic function. The mutation His328→Asn caused the essential loss in activity, while the mutation His103→Asn yielded a mutant enzyme that retained 59% of the κ₀/K_m of that for the wild-type enzyme. Since mutants of other α-amylases acting on α-1,4-glucosidic bond linkage lost most of their activity by the site-directed mutagenesis at their equivalent residues to His103 and His328, the retaining of activity by Hisl03→Asn mutation in B. cereus oligo-1,6-glucosidase revealed the distinguished role of His103 for the hydrolysis of α-1,6-glucosidic bond linkage.     

An analysis of reaction pathways for proton tunnelling in methylamine dehydrogenase

Computational methods have now become a valuable tool to understand the way in which enzymes catalyse chemical reactions and to aid the interpretation of a diverse set of experimental data. This study focuses on the influence of the condensed-phase environment structure on proton transfer mechanisms, with an aim to understand how C-H bond cleavage is mediated in enzymatic reactions. We shall use a combination of molecular simulation, ab initio or semi-empirical quantum chemistry and semi-classical multidimensional tunnelling methods to consider the primary kinetic isotope effects of the enzyme methylamine dehydrogenase (MADH), with reference to an analogous application to triosephosphate isomerase. Analysis of potentially reactive conformations of the system, and correlation with experimental isotope effects, have highlighted that a quantum tunnelling mechanism in MADH may be modulated by specific amino acid residues, such as Asp428, Thr474 and Asp384.     

Probing domain mobility in a flavocytochrome

The crystal structures of various different members of the family of fumarate reductases and succinate dehydrogenases have allowed the identification of a mobile clamp (or capping) domain [e.g., Taylor, P., Pealing, S. L., Reid, G. A., Chapman, S. K., and Walkinshaw, M. D. (1999) Nat. Struct. Biol. 6, 1108-1112], which has been proposed to be involved in regulating accessibility of the active site to substrate. To investigate this, we have constructed the A251C:S430C double mutant form of the soluble flavocytochrome c(3) fumarate reductase from Shewanella frigidimarina, to introduce an interdomain disulfide bond between the FAD-binding and clamp domains of the enzyme, thus restricting relative mobility between the two. Here, we describe the kinetic and crystallographic analysis of this double mutant enzyme. The 1.6 A resolution crystal structure of the A251C:S430C enzyme under oxidizing conditions reveals the formation of a disulfide bond, while Ellman analysis confirms its presence in the enzyme in solution. Kinetic analyses with the enzyme in both the nonbridged (free thiol) and the disulfide-bridged states indicate a slight decrease in the rate of fumarate reduction when the disulfide bridge is present, while solvent-kinetic-isotope studies indicate that in both wild-type and mutant enzymes the reaction is rate limited by proton and/or hydride transfer during catalysis. The limited effects of the inhibition of clamp domain mobility upon the catalytic reaction would indicate that such mobility is not essential for the regulation of substrate access or product release.     

Studies of specificity and catalysis in trypsin by structural analysis of site-directed mutants

We are probing the determinants of catalytic function and substrate specificity in serine proteases by kinetic and crystallographic characterization of genetically engineered site-directed mutants of rat trypsin. The role of the aspartyl residue at position 102, common to all members of the serine protease family, has been tested by substitution with asparagine. In the native enzyme, Asp102 accepts a hydrogen bond from the catalytic base His57, which facilitates the transfer of a proton from the enzyme nucleophile Ser195 to the substrate leaving group. At neutral pH, the mutant is four orders of magnitude less active than the naturally occurring enzyme, but its binding affinity for model substrates is virtually undiminished. Crystallographic analysis reveals that Asn102 donates a hydrogen bond to His57, forcing it to act as donor to Ser195. Below pH 6, His57 becomes statistically disordered. Presumably, the di-protonated population of histidyl side chains are unable to hydrogen bond to Asn102. Steric conflict may cause His57 to rotate away from the catalytic site. These results suggest that Asp102 not only provides inductive and orientation effects, but also stabilizes the productive tautomer of His57. Three experiments were carried out to alter the substrate specificity of trypsin. Glycine residues at positions 216 and 226 in the substrate-binding cavity were replaced by alanine residues in order to differentially affect lysine and arginine substrate binding. While the rate of catalysis by the mutant enzymes was reduced in the mutant enzymes, their substrate specificity was enhanced relative to trypsin. The increased specificity was caused by differential effects on the catalytic activity towards arginine and lysine substrates. The Gly----Ala substitution at 226 resulted in an altered conformation of the enzyme which is converted to an active trypsin-like conformation upon binding of a substrate analog. In a third experiment, Lys189, at the bottom of the specificity pocket, was replaced with an aspartate with the expectation that specificity of the enzyme might shift to aspartate. The mutant enzyme is not capable of cleaving at Arg and Lys or Asp, but shows an enhanced chymotrypsin-like specificity. Structural investigations of these mutants are in progress.