Purification and properties of the P2 primary alkylsulphohydrolase of the detergent-degrading bacterium pseudomonas C12B.

The P2 primary alkylsulphohydrolase of the soil bacterium Pseudomonas C12B was purified to homogeneity (200-250-fold) by column chromatography on DEAE-cellulose, Sephadex G-100 and butyl-agarose. The intact protein is a dimer with a mol. wt. of 160 000. Activity towards primary alkyl sulphate esters was maximal at pH 8.3, varied little in the range pH 7.8-8.7, but decreased sharply at higher pH. For a homologous series of primary alkyl sulphate substrates (C6-C12), logKm decreased linearly with increasing chain length, corresponding to a contribution to the free energy of association between enzyme and substrate of -2.5kJ/mol for each additional CH2 group in the alkyl chain. logKi for the competitive inhibition by secondary alkyl 2-sulphate esters followed a similar pattern (-2.4kJ/mol for each additional CH2 group) except that only n-1 carbon atoms effectively participate in hydrophobic bonding, implying that the C-1 methyl group is not involved. logKi values for inhibition primary alkanesulphonates also depended linearly on chain length but with a diminished gradient, indicating a free-energy increment of -1.2kJ/mol per additional CH2 group. The collective results showed the presence of a hydrophobic site on the enzyme capable of accomodating an alkyl chain of considerable length. Cationic structures (in the form of arginine, lysine or histidine), whose presence might be expected for binding the anionic sulphate group, were not detectable at the active site.     

Kinetic and crystallographic analysis of active site mutants of Escherichia coli gamma-aminobutyrate aminotransferase

The E. coli isozyme of gamma-aminobutyrate aminotransferase (GABA-AT) is a tetrameric pyridoxal phosphate-dependent enzyme that catalyzes transamination between primary amines and alpha-keto acids. The roles of the active site residues V241, E211, and I50 in the GABA-AT mechanism have been probed by site-directed mutagenesis. The beta-branched side chain of V241 facilitates formation of external aldimine intermediates with primary amine substrates, while E211 provides charge compensation of R398 selectively in the primary amine half-reaction and I50 forms a hydrophobic lid at the top of the substrate binding site. The structures of the I50Q, V241A, and E211S mutants were solved by X-ray crystallography to resolutions of 2.1, 2.5, and 2.52 A, respectively. The structure of GABA-AT is similar in overall fold and active site structure to that of dialkylglycine decarboxylase, which catalyzes both transamination and decarboxylation half-reactions in its normal catalytic cycle. Therefore, an attempt was made to convert GABA-AT into a decarboxylation-dependent aminotransferase similar to dialkylglycine decarboxylase by systematic mutation of E. coli GABA-AT active site residues. Two of the twelve mutants presented, E211S/I50G/C77K and E211S/I50H/V80D, have approximately 10-fold higher decarboxylation activities than the wild-type enzyme, and the E211S/I50H/V80D has formally changed the reaction specificity to that of a decarboxylase.     

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.     

Inhibition of the aminopeptidase from Aeromonas proteolytica by aliphatic alcohols. Characterization of the hydrophobic substrate recognition site.

Seven aliphatic and two aromatic alcohols were tested as reporters of the substrate selectivity of the aminopeptidase from Aeromonas proteolytica (AAP). This series of alcohols was chosen to systematically probe the effect of carbon chain length, steric bulk, and inhibitor shape on the inhibition of AAP. Initially, however, the question of whether AAP is denatured in the presence of aliphatic alcohols was addressed. On the basis of circular dichroism (CD), electronic absorption, and fluorescence spectra, the secondary structure of AAP, with and without added aliphatic alcohols, was unchanged. These data clearly indicate that AAP is not denatured in aliphatic alcohols, even up to concentrations of 20% (v/v). All of the alcohols studied were competitive inhibitors of AAP with K(i) values between 860 and 0.98 mM. The clear trend in the data was that as the carbon chain length increases from one to four, the K(i) values increase. Branching of the carbon chains also increases the K(i) values, but large bulky groups, such as that found in tert-butyl alcohol, do not inhibit AAP as well as leucine analogues, such as 3-methyl-1-butanol. The competitive nature of the inhibition indicates that the substrate and each alcohol studied are mutually exclusive due to binding at the same site on the enzyme. On the basis of EPR and electronic absorption data for Co(II)-substituted AAP, none of the alcohols studied binds to the dinuclear metallo-active site of AAP. Thus, reaction of the inhibitory alcohols with the catalytic metal ions cannot constitute the mechanism of inhibition. Combination of these data suggests that each of these inhibitors bind only to the hydrophobic pocket of AAP and, consequently, block the binding of substrate. Thus, the first step in peptide hydrolysis is the recognition of the N-terminal amino acid side chain by the hydrophobic pocket adjacent to the dinuclear active site of AAP.     

Thermodynamic basis of resistance to HIV-1 protease inhibition: calorimetric analysis of the V82F/I84V active site resistant mutant

One of the most serious side effects associated with the therapy of HIV-1 infection is the appearance of viral strains that exhibit resistance to protease inhibitors. The active site mutant V82F/I84V has been shown to lower the binding affinity of protease inhibitors in clinical use. To identify the origin of this effect, we have investigated the binding thermodynamics of the protease inhibitors indinavir, ritonavir, saquinavir, and nelfinavir to the wild-type HIV-1 protease and to the V82F/I84V resistant mutant. The main driving force for the binding of all four inhibitors is a large positive entropy change originating from the burial of a significant hydrophobic surface upon binding. At 25 degrees C, the binding enthalpy is unfavorable for all inhibitors except ritonavir, for which it is slightly favorable (-2.3 kcal/mol). Since the inhibitors are preshaped to the geometry of the binding site, their conformational entropy loss upon binding is small, a property that contributes to their high binding affinity. The V82F/I84V active site mutation lowers the affinity of the inhibitors by making the binding enthalpy more positive and making the entropy change slightly less favorable. The effect on the enthalpy change is, however, the major one. The predominantly enthalpic effect of the V82F/I84V mutation is consistent with the idea that the introduction of the bulkier Phe side chain at position 82 and the Val side chain at position 84 distort the binding site and weaken van der Waals and other favorable interactions with inhibitors preshaped to the wild-type binding site. Another contribution of the V82F/I84V to binding affinity originates from an increase in the energy penalty associated with the conformational change of the protease upon binding. The V82F/I84V mutant is structurally more stable than the wild-type protease by about 1.4 kcal/mol. This effect, however, affects equally the binding affinity of substrate and inhibitors.     

Rat butyrylcholinesterase-catalysed hydrolysis of N-alkyl homologues of benzoylcholine

The purpose of this work was to study the catalytic properties of rat butyrylcholinesterase with benzoylcholine (BzCh) and N-alkyl derivatives of BzCh (BCHn) as substrates. Complex hysteretic behaviour was observed in the approach to steady-state kinetics for each ester. Hysteresis consisted of a long lag phase with damped oscillation. The presence of a long lag phase, with no oscillations, in substrate hydrolysis by rat butyrylcholinesterase was also observed with N-methylindoxyl acetate as substrate. Hysteretic behaviour was explained by the existence of two interconvertible butyrylcholinesterase forms in slow equilibrium, while just one of them is catalytically active. The damped oscillations were explained by the existence of different substrate conformational states and/or aggregates (micelles) in slow equilibrium. Different substrate conformational states were confirmed by 1H-NMR. The K(m) values for substrates decreased as the length of the alkyl chain increased. High affinity of the enzyme for the longest alkyl chain length substrates was explained by multiple hydrophobic interactions of the alkyl chain with amino acid residues lining the active site gorge. Molecular modelling studies supported this interpretation; docking energy decreased as the length of the alkyl chain increased. The long-chain substrates had reduced k(cat) values. Docking studies showed that long-chain substrates were not optimally oriented in the active site for catalysis, thus explaining the slow rate of hydrolysis. The hydrolytic rate of BCH12 and longer alkyl chain esters vs. substrate concentration showed a premature plateau far below V(max). This was due to the loss of substrate availability. The best substrates for rat butyrylcholinesterase were short alkyl homologues, BzCh - BCH4.     

Characterization of kinetics and thermostability of Acremonium strictum glucooligosaccharide oxidase

The kinetic and thermostability properties of a glucooligosaccharide oxidase from Acremonium strictum were determined. This enzyme produces only maltobionic acid from maltose. It is most active at pH 9 to 10.5, and is most stable at pH 6.5. Values of both K(M) and V(max) on maltose are highest at pH 10. The highest values of K(M) and V(max) occur with glucose, maltopentaose, and maltoheptaose, whereas the lowest values of K(M) are with maltotriose and of V(max) are with maltohexaose. Values of K(M) with any substrate and at any pH are always substantially above 1 mM. Activation energies for catalysis and thermoinactivation are 23 kJ/mol and 421 kJ/mol, respectively. The N-terminal sequence is not homologous with any other oxidase, but has some homology with other proteins having different functions. These unusual properties suggest that glucooligosaccharides may not be the primary substrates of this enzyme.     

N-alkanamines as substrates to probe the hydrophobic region of bovine serum amine oxidase active site: a kinetic and spectroscopic study

Kinetic and spectroscopic studies were carried out to study the role of hydrophobic effect on the activity of bovine serum amine oxidase (BSAO). Increasing the chain length of the substrates (linear aliphatic primary monoamines), the affinity for the active site increases while the catalytic constant decreases in accordance with a relative low value of dielectric constant (about 10) estimated for the microenvironment of BSAO active site using a fluorescent probe sensitive to solvent polarity. The aliphatic chain of 1-aminononane induces a shift in the pK(a) of the product Schiff base, the hydrolysis of which appears to be a rate-determining step of the reaction. Furthermore, circular dichroism studies highlighted the "flexibility" of BSAO secondary structure that can explain the wide substrate specificity of this enzyme. These results should be useful to elucidate the substrate/inhibitor preferences of CuAOs, in particular of the human enzyme.     

Mechanism of nitroalkane oxidase: 2. pH and kinetic isotope effects

Nitroalkane oxidase catalyzes the oxidation of nitroalkanes to aldehydes or ketones with production of nitrite and hydrogen peroxide. pH and kinetic isotope effects with [1, 1-(2)H(2)]nitroethane have been used to study the mechanism of this enzyme. The V/K(ne) pH profile is bell-shaped. A group with a pK(a) value of about 7 must be unprotonated and one with a pK(a) value of 9.5 must be protonated for catalysis. The lower pK(a) value is seen also in the pK(is) profile for the competitive inhibitor valerate, indicating that nitroethane has no significant external commitments to catalysis. The (D)(V/K)(ne) value is pH-independent with a value of 7.5, whereas the (D)V(max) value increases from 1.4 at pH 8.2 to a limiting value of 7.4 below pH 5. The V(max) pH profile decreases at low and high pH, with pK(a) values of 6.6 and 9.5, respectively. Imidazole, which activates the enzyme, affects the V(max) but not the V/K(ne) pH profile. In the presence of imidazole at pH 7 the (D)V(max) value increases to a value close to the intrinsic value, consistent with cleavage of the carbon-hydrogen bond of the substrate being fully rate-limiting for catalysis in the presence of imidazole.     

Tissue transglutaminase acylation: Proposed role of conserved active site Tyr and Trp residues revealed by molecular modeling of peptide substrate binding

Transglutaminases (TGases) catalyze the cross-linking of peptides and proteins by the formation of gamma-glutamyl-epsilon-lysyl bonds. Given the implication of tissue TGase in various physiological disorders, development of specific tissue TGase inhibitors is of current interest. To aid in the design of peptide-based inhibitors, a better understanding of the mode of binding of model peptide substrates to the enzyme is required. Using a combined kinetic/molecular modeling approach, we have generated a model for the binding of small acyl-donor peptide substrates to tissue TGase from red sea bream. Kinetic analysis of various N-terminally derivatized Gln-Xaa peptides has demonstrated that many CBz-Gln-Xaa peptides are typical in vitro substrates with K(M) values between 1.9 mM and 9.4 mM, whereas Boc-Gln-Gly is not a substrate, demonstrating the importance of the CBz group for recognition. Our binding model of CBz-Gln-Gly on tissue TGase has allowed us to propose the following steps in the acylation of tissue TGase. First, the active site is opened by displacement of conserved W329. Second, the substrate Gln side chain enters the active site and is stabilized by hydrophobic interaction with conserved residue W236. Third, a hydrogen bond network is formed between the substrate Gln side chain and conserved residues Y515 and the acid-base catalyst H332 that helps to orient and activate the gamma-carboxamide group for nucleophilic attack by the catalytic sulphur atom. Finally, an H-bond with Y515 stabilizes the oxyanion formed during the reaction.     

Substrate specificity of king cobra (Ophiophagus hannah) venom L-amino acid oxidase

1. Substrate specificity of purified king cobra (Ophiophagus hannah) venom L-amino acid oxidase was investigated. 2. The enzyme was highly specific for the L-enantiomer of amino acid. Effective oxidation of L-amino acid by the enzyme requires the presence of a free primary alpha-amino group but the alpha-carboxylate group is not as critical for the catalysis. 3. The enzyme was very active against L-Lys, L-Phe, L-Leu, L-Tyr, L-Tryp, L-Arg, L-Met, L-ornithine, L-norleucine and L-norvaline and moderately active against L-His, L-cystine and L-Ileu. Other L-amino acids were oxidized slowly or not oxidized. 4. The data suggest the presence of a side chain binding site in the enzyme, and that the binding site comprises at least five 'subsites': the hydrophobic subsites a, b and c; and the two 'amino' binding subsites d and e. Subsite b appears to be able to accommodate two methylene/methyl carbons.     

Probing for a hydrophobic a binding register in prostate-specific membrane antigen with phenylalkylphosphonamidates

To explore for the existence of an auxiliary hydrophobic binding register remote from the active site of PSMA a series of phenylalkylphosphonamidate derivatives of glutamic acid were synthesized and evaluated for their inhibitory potencies against PSMA. Both the phenyl- and benzylphosphonamidates (1a and 1b) exhibited only modest inhibitory potency against. The phenethyl analog 1c was intermediate in inhibitory potency while inhibitors possessing a longer alkyl tether from the phenyl ring, resulted in markedly improved K(i) values. The greatest inhibitory potency was obtained for the inhibitors in which the phenyl ring was extended furthest from the central phosphorus (1f, n=5 and 1g, n=6). The slightly serrated pattern that emerged as the alkyl tether increased from three to six methylene units suggests that inhibitory potency is not simply correlated to increased hydrophobicity imparted by the phenylalkyl chain, but rather that one or more hydrophobic binding registers may exist remote from the substrate recognition architecture in the active site of PSMA.     

pH and deuterium kinetic isotope effects studies on the oxidation of choline to betaine-aldehyde catalyzed by choline oxidase

The FAD-dependent choline oxidase catalyzes the four-electron oxidation of choline to glycine-betaine, with betaine-aldehyde as intermediate. The enzyme is capable of accepting either choline or betaine-aldehyde as a substrate, allowing the investigation of the reaction mechanism for both the conversion of choline to betaine-aldehyde and of betaine-aldehyde to glycine-betaine. In the present study, pH and deuterium kinetic isotope effects with [1,2-2H(4)]-choline were used to study the mechanism of oxidation of choline to betaine-aldehyde. The V/K and V(max) pH-profiles increased to limiting values with increasing pH, suggesting the presence of a catalytic base essential for catalysis at the enzyme active site. From the V/K pH-profile with [1,2-2H(4)]-choline, a pK(a) of 8.0 was determined for the catalytic base. This pK(a) was shifted to 7.5 in the V/K pH-profile with choline, indicating a significant commitment to catalysis with this substrate. In agreement with this conclusion, the D(V/K) values decreased from a limiting value of 12.4 below pH 6.5 to a limiting value of 4.1 above pH 9.5. The large D(V/K) values at low pH are consistent with carbon-hydrogen bond cleavage of choline being nearly irreversible and fully rate-limiting at low pH. Based on comparison of amino acid sequences and previous structural and mechanistic studies on other members of the GMC oxidoreductase superfamily, the identity of the catalytic base of choline oxidase is proposed.     

The role of tyrosine 177 in human 11beta-hydroxysteroid dehydrogenase type 1 in substrate and inhibitor binding: an unlikely hydrogen bond donor for the substrate

The catalytic motif (YSASK) at the active site of 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) is conserved across different species. The crystal structures of the human, guinea pig and mouse enzymes have been resolved to help identify the non-conserved residues at the active site. A tyrosine residue (Y177) upstream of the catalytic motif in human 11beta-HSD1 represents the largest difference at the active sites between the human and the rodent enzyme where the corresponding residue is glutamine. Although Y177 was postulated as a potential hydrogen bond donor in substrate binding in crystal structure-based modeling, no experimental evidence is available to support this notion. Here, we report that Y177 is not a hydrogen bond donor in substrate binding because removal of the hydroxyl group from its side chain by mutagenesis (Y177F) did not significantly change the Km value for cortisone. However, removal of the hydrophobic side chain by changing tyrosine to alanine (Y177A) or substitution with a hydrophilic side chain by changing tyrosine to glutamine (Y177Q) increased Km values for cortisone. These data suggest that Y177 is involved in substrate binding through its hydrophobic side chain but not by hydrogen bonding. In addition, the three mutations had little effect on the binding of the rodent substrate 11-dehydrocorticosterone, suggesting that Y177 does not confer substrate specificity. However, the same mutations reduced the affinity of the licorice derived 11beta-HSD1 inhibitor glycyrrhetinic acid by about 6- to 10-fold. Interestingly, the affinity of carbenoxolone, the hemisuccinate ester of glycyrrhetinic acid with a similar potency against the wildtype enzyme, was not drastically affected by the same mutations at Y177. These data suggest that Y177 has a unique role in inhibitor binding. Molecular modeling with glycyrrhetinic acid led to findings consistent with the experimental data and provided potential interaction mechanisms. Our data suggest that Y177 plays an important role in both substrate and inhibitor binding but it is unlikely a hydrogen bond donor for the substrate.     

Inhibitory effects of anions and active site amino acid sequence of chicken liver L-2-hydroxyacid oxidase A, a member of the FMN-dependent α-hydroxyacid oxidizing enzyme family

Monocarboxylic acids with aliphatic chains were found to be mixed inhibitors of chicken liver L-2-hydroxyacid oxidase A when L-2-hydroxy-4-methylthiobutanoic acid was used as the substrate. The finding that the binding affinity of the enzyme for monocarboxylic acids was directly proportional to the number of carbon atoms in the chain strongly suggests that in addition to the electrostatic interaction due to the carboxyl moiety, hydrophobic forces may also be involved in the binding affinity of monocarboxylic acids to the enzyme's active site. Oxalate, a dicarboxylic acid, also resulted in a mixed-type inhibition of chicken liver L-2-hydroxyacid oxidase A, and, surprisingly, its binding affinity to the enzyme was found to be quite high as compared with monocarboxylic acids. This is probably due to the fact that the two carboxyl groups of oxalate give rise to electrostatic interactions with the positively charged side chains of two adjacent residues in the polypeptide chain. The inhibitory effects of other dicarboxylic acids was found to decrease as the number of carbon atoms in the chain increased. Oxamate was found however to be a novel type of potent inhibitor of the enzyme. All in all, these kinetic studies and the amino acid sequence determination in the active site region after limited proteolysis of the polypeptide chain definitely establish that chicken liver NADH/FMN containing L-2-hydroxyacid oxidase A is a member of the FMN-dependent α-hydroxyacid oxidizing enzyme family.     

Involvement of a flavosemiquinone in the enzymatic oxidation of nitroalkanes catalyzed by 2-nitropropane dioxygenase

2-Nitropropane dioxygenase (EC 1.13.11.32) catalyzes the oxidation of nitroalkanes into their corresponding carbonyl compounds and nitrite. In this study, the ncd-2 gene encoding for the enzyme in Neurospora crassa was cloned, expressed in Escherichia coli, and the resulting enzyme was purified. Size exclusion chromatography, heat denaturation, and mass spectroscopic analyses showed that 2-nitropropane dioxygenase is a homodimer of 80 kDa, containing a mole of non-covalently bound FMN per mole of subunit, and is devoid of iron. With neutral nitroalkanes and anionic nitronates other than propyl-1- and propyl-2-nitronate, for which a non-enzymatic free radical reaction involving superoxide was established using superoxide dismutase, substrate oxidation occurs within the enzyme active site. The enzyme was more specific for nitronates than nitroalkanes, as suggested by the second order rate constant k(cat)/K(m) determined with 2-nitropropane and primary nitroalkanes with alkyl chain lengths between 2 and 6 carbons. The steady state kinetic mechanism with 2-nitropropane, nitroethane, nitrobutane, and nitrohexane, in either the neutral or anionic form, was determined to be sequential, consistent with oxygen reacting with a reduced form of enzyme before release of the carbonyl product. Enzyme-monitored turnover with ethyl nitronate as substrate indicated that the catalytically relevant reduced form of enzyme is an anionic flavin semiquinone, whose formation requires the substrate, but not molecular oxygen, as suggested by anaerobic substrate reduction with nitroethane or ethyl nitronate. Substrate deuterium kinetic isotope effects with 1,2-[(2)H(4)]nitroethane and 1,1,2-[(2)H(3) ethyl nitronate at pH 8 yielded normal and inverse effects on the k(cat)/K(m) value, respectively, and were negligible on the k(cat) value. The k(cat)/K(m) and k(cat) pH profiles with anionic nitronates showed the requirement of an acid, whereas those for neutral nitroalkanes were consistent with the involvement of both an acid and a base in catalysis. The kinetic data reported herein are consistent with an oxidasestyle catalytic mechanism for 2-nitropropane dioxygenase, in which the flavin-mediated oxidation of the anionic nitronates or neutral nitroalkanes and the subsequent oxidation of the enzyme-bound flavin occur in two independent steps.     

Conversion of methylamine dehydrogenase to a long-chain amine dehydrogenase by mutagenesis of a single residue

Methylamine dehydrogenase (MADH) is a tryptophan tryptophylquinone (TTQ) dependent enzyme that catalyzes the oxidative deamination of primary amines. Amino acid residues of both the TTQ-bearing beta subunit and the noncatalytic alpha subunit line a substrate channel that leads from the protein surface to the enzyme active site. Phe55 of the alpha subunit is located at the opening of the active site. Conversion of alphaPhe55 to alanine dramatically alters the substrate preference of MADH. The K(m) for methylamine increases from 9 microM to 15 mM. The preferred substrates are now primary amines with chain lengths of at least seven carbons. The K(m) for 1, 10-diaminodecane is 11 microM, compared to 1.2 mM for wild-type MADH. Despite the large variation in K(m) values, k(cat) values are relatively unaffected by the mutation. Molecular modeling of substrates into the crystal structure of the enzyme active site and substrate channel provides an explanation for the dramatic changes in substrate specificity caused by this mutation of a single amino acid residue.     

Human dihydrofolate reductase: reduction of alternative substrates, pH effects, and inhibition by deazafolates.

The kinetics of the NADPH-dependent reduction of 7,8-dihydrofolate, folate, and 7,8-dihydrobiopterin by human dihydrofolate reductase have been examined over the pH range from 4.0 to 9.5. The V and V/K profiles obtained with the three substrates indicate that a single ionizing residue at the active site of the enzyme must be protonated for catalysis. Both the maximum velocity of the reactions and the rate of interaction of the substrates with the enzyme-NADPH complex decrease in the order dihydrofolate greater than dihydrobiopterin much greater than folate. From the pK values of the V/K profiles, it can be concluded that, while dihydrofolate behaves as a sticky substrate and dihydrobiopterin exhibits slight stickiness, folate is not a sticky substrate. Further support for this conclusion comes from the results of deuterium isotope effects. The pK values obtained from both the V and V/Kfolate profiles are similar to the intrinsic pK value of 5.6 for both the free enzyme and the enzyme-NADPH complex. The folate analogue, 5-deazafolate, is not a substrate, but it undergoes strong interaction with the enzyme. This interaction, which is enhanced by the presence of NADPH, is due to protonation of the bound ligand that does not involve the single ionizing group at the active center of the enzyme. Difference spectra yield evidence for the protonation of bound 5-deazafolate and show that, on binding to the enzyme-NADPH complex, the pK of the N-8 atom is raised to about 10 from a value of about 4 in solution. The results are in accord with those of a recent paper on the three-dimensional structure of the enzyme-5-deazafolate complex [Davies, J.F., Delcamp, T.J., Prendergast, N.J., Ashfors, V.A., Freisheim, J.H., & Kraut, J. (1990) Biochemistry 29, 9467-9479] which indicate that there is hydrogen bond formation between N-8 of the ligand and the carbonyl group of Ile-7. However, the present findings do not support the idea that bound 5-deazafolate resembles the transition-state complex for folate reduction. Quinazolines also interact strongly with the enzyme but in a pH-independent manner. The dissociation constants for the binary complexes are an order of magnitude lower than that for the binding to the enzyme of unprotonated 5-deazafolate. This difference reflects the hydrophobic nature of the amino acid residues at the active site that are near the N-5 and N-8 nitrogens of bound pterins.     

Active site residue involvement in monoamine or diamine oxidation catalysed by pea seedling amine oxidase

The structures of copper amine oxidases from various sources show good similarity, suggesting similar catalytic mechanisms for all members of this enzyme family. However, the optimal substrates for each member differ, depending on the source of the enzyme and its location. The structural factors underlying substrate selectivity still remain to be discovered. With this in view, we examined the kinetic behaviour of pea seedling amine oxidase with cadaverine and hexylamine, the first bearing two, and the second only one, positively charged amino group. The dependence of K(m) and catalytic constant (k(c)) values on pH, ionic strength and temperature indicates that binding of the monoamine is driven by hydrophobic interactions. Instead, binding of the diamine is strongly facilitated by electrostatic factors, controlled by polar side-chains and two titratable residues present in the active site. The position of the docked substrate is also essential for the participation of titratable amino acid residues in the following catalytic steps. A new mechanistic model explaining the substrate-dependent kinetics of the reaction is discussed.     

Alteration of substrate specificity of leucine dehydrogenase by site-directed mutagenesis

The residues L40, A113, V291, and V294, in leucine dehydrogenase (LeuDH), predicted to be involved in recognition of the substrate side chain, have been mutated on the basis of the molecular modeling to mimic the substrate specificities of phenylalanine (PheDH), glutamate (GluDH), and lysine dehydrogenases (LysDH). The A113G and A113G/V291L mutants, imitating the PheDH active site, displayed activities toward -phenylalanine and phenylpyruvate with 1.6 and 7.8% of k_cat values of the wild-type enzyme for the preferred substrates, -leucine and its keto-analog, respectively. Indeed, the residue A113, corresponding to G114 in PheDH, affects the volume of the side-chain binding pocket and has a critical role in discrimination of the bulkiness of the side chain. Another two sets of mutants, substituting L40 and V294 of LeuDH with the corresponding residues predicted in GluDH and LysDH, were also constructed and characterized. Emergence of GluDH and LysDH activities in L40K/V294S and L40D/V294S mutants, respectively, indicates that the two corresponding residues in the active site of amino acid dehydrogenases are important for discrimination of the hydrophobicity/polarity of the aliphatic substrate side chain. All these results demonstrate that the substrate specificities of the amino acid dehydrogenases can be altered by protein engineering. The engineered dehydrogenases are expected to be used for production and detection of natural and non-natural amino acids.