Serine hydroxymethyltransferase: mechanism of the racemization and transamination of D- and L-alanine

The reaction specificity and stereochemical control of Escherichia coli serine hydroxymethyltransferase were investigated with D- and L-alanine as substrates. An active-site H228N mutant enzyme binds both D- and L-alanine with Kd values of 5 mM as compared to 30 and 10 mM, respectively, for the wild-type enzyme. Both wild-type and H228N enzymes form quinonoid complexes absorbing at 505 nm by catalyzing the loss of the alpha-proton from both D- and L-alanine. Racemization and transamination reactions were observed to occur with both alanine isomers as substrates. The relative rates of these reactions are quinonoid formation greater than alpha-proton solvent exchange greater than racemization greater than transamination. The observation that the rate of quinonoid formation with either alanine isomer is an order of magnitude faster than solvent exchange suggests that the alpha-protons from both D- and L-alanine are transferred to base(s) on the enzyme. The rate of racemization is 2 orders of magnitude slower than the formation of the quinonoid complexes. This latter difference in rate suggests that the quinonoid complexes formed from D- and L-alanine are not identical. The difference in structure of the two quinonoid complexes is proposed to be the active-site location of the alpha-protons lost from the two alanine isomers, rather than two orientations of the pyridoxal phosphate ring. The results are consistent with a two-base mechanism for racemization.     

Kinetic studies of formate dehydrogenase

1. The kinetic mechanism of formate dehydrogenase is a sequential pathway. 2. The binding of the substrates proceeds in an obligatory order, NAD(+) binding first, followed by formate. 3. It seems most likely that the interconversion of the central ternary complex is extremely rapid, and that the rate-limiting step is the formation or possible isomerization of the enzyme-coenzyme complexes. 4. The secondary plots of the inhibitions with HCO(3) (-) and NO(3) (-) are non-linear, which suggests that more than one molecule of each species is able to bind to the same enzyme form. 5. The rate of the reverse reaction with carbon dioxide at pH6.0 is 20 times that with bicarbonate at pH8.0, although no product inhibition could be detected with carbon dioxide. The low rate of the reverse reaction precluded any steady-state analysis as the enzyme concentrations needed to obtain a measurable rate are of the same order as the K(m) values for NAD(+) and NADH.     

Selective proteolysis of cytosolic aspartate aminotransferase by a new microbial protease

A protease from Streptomyces violaceochromogenes (Murao, S., Nishino, Y., & Maeda, Y. (1984) Agric. Biol. Chem. 48, 2163-2166) is known to inactivate pig heart aspartate aminotransferase [EC 2.6.1.1]. Chemical analysis of the core proteins and peptide fragments produced upon proteolysis of the aminotransferase revealed that peptide bond cleavage occurred specifically at Leu 20 with concomitant inactivation. Neither inactivation nor peptide bond cleavage was observed with the mitochondrial isoenzyme. The proteolytically produced derivative 21-412 of the cytosolic isoenzyme retained approximately 0.1% enzymic activity for transamination with natural dicarboxylic substrates. The pyridoxal form of the derivative 21-412 was fully converted by cysteinesulfinate or alanine to the pyridoxamine form and conversely the pyridoxamine form of the derivative was also fully converted by 2-oxoglutarate or pyruvate into the pyridoxal form, indicating that the derivative was still catalytically competent. However, the rates of reaction with dicarboxylic substrates were much reduced whereas the rates with monocarboxylic substrates remained at an order of magnitude similar to that observed with the native enzyme. Thus the NH2-terminal segment appears to be an import structural component which determines the substrate specificity of aspartate aminotransferase for dicarboxylic keto and amino acids. A substantial alteration in the molecular structure accompanying the loss of the NH2-terminal 20 residues was also reflected by the decrease in heat stability and in the lowering of the pKa value for His 68, which is involved in the intersubunit interaction of this dimeric enzyme.     

The origin of reaction specificity in serine hydroxymethyltransferase

Cytosolic serine hydroxymethyltransferase has been shown previously to exhibit both broad substrate and reaction specificity. In addition to cleaving many different 3-hydroxyamino acids to glycine and an aldehyde, the enzyme also catalyzes with several amino acid substrate analogs decarboxylation, transamination, and racemization reactions. To elucidate the relationship of the structure of the substrate to reaction specificity, the interaction of both amino acid and folate substrates and substrate analogs with the enzyme has been studied by three different methods. These methods include investigating the effects of substrates and substrate analogs on the thermal denaturation properties of the enzyme by differential scanning calorimetry, determining the rate of peptide hydrogen exchange with solvent protons, and measuring the optical activity of the active site pyridoxal phosphate. All three methods suggest that the enzyme exists as an equilibrium between "open" and "closed" forms. Amino acid substrates enter and leave the active site in the open form, but catalysis occurs in the closed form. The data suggest that the amino acid analogs that undergo alternate reactions, such as racemization and transamination, bind only to the open form of the enzyme and that the alternate reactions occur in the open form. Therefore, one role for forming the closed form of the enzyme is to block side reactions and confer reaction specificity.     

Anomalous “saturation kinetics” occurring in the reaction of l-α-aminoadipate with pig heart aspartate aminotransferase

Low concentrations of l-α-aminoadipate and α-ketoadipate do not form stable complexes with the phosphopyridoxal and phosphopyridoxamine forms of pig heart aspartate aminotransferase, respectively, as judged by direct spectroscopic analyses. Furthermore, the addition of 40 mm aminoadipate did not significantly inhibit the transamination reaction between aspartate and α-ketoglutarate. Nevertheless, aminoadipate complexes must be formed, albeit in very small amounts, because the phosphopyridoxal form of the enzyme undergoes transamination with aminoadipate to form the phosphopyridoxamine form and α-ketoadipate. Since the reaction of aminoadipate with the phosphopyridoxal form of the enzyme shows apparent saturation kinetics at substrate concentrations that are insufficient to form detectable intermediary complexes, the rate-limiting step in the reaction must be a slow transition of the enzyme occurring prior to the amino acid interaction. The maximum rate of this transamination reaction decreased with an increase in the buffer anion concentration; the anion must, therefore, inhibit the slow enzyme transition. The experimental data are consistent with a simple model in which the anion dissociates before the slow transition that allows the aminoadipate to react. The turnover rate of aminoadipate is approximately three orders of magnitude less than those of both aspartate and glutamate. The latter substrates do not appear, therefore, to require the slow enzyme transition that is necessary for aminoadipate to react.     

Rapid kinetic and isotopic studies on dialkylglycine decarboxylase

The two half-reactions of the pyridoxal 5'-phosphate (PLP)-dependent enzyme dialkylglycine decarboxylase (DGD) were studied individually by multiwavelength stopped-flow spectroscopy. Biphasic behavior was found for the reactions of DGD-PLP, consistent with two coexisting conformations observed in steady-state kinetics [Zhou, X., and Toney, M. D. (1998) Biochemistry 37, 5761--5769]. The half-reaction kinetic parameters depend on alkali metal ion size in a manner similar to that observed for steady-state kinetic parameters. The fast phase maximal rate constant for the 2-aminoisobutyrate (AIB) decarboxylation half-reaction with the potassium form of DGD-PLP is 25 s(-1), while that for the transamination half-reaction between DGD-PMP and pyruvate is 75 s(-1). The maximal rate constant for the transamination half-reaction of the potassium form of DGD-PLP with L-alanine is 24 s(-1). The spectral data indicate that external aldimine formation with either AIB or L-alanine and DGD-PLP is a rapid equilibrium process, as is ketimine formation from DGD-PMP and pyruvate. Absorption ascribable to the quinonoid intermediate is not observed in the AIB decarboxylation half-reaction, but is observed in the dead-time of the stopped-flow in the L-alanine transamination half-reaction. The [1-(13)C]AIB kinetic isotope effect (KIE) on k(cat) for the steady-state reaction is 1.043 +/- 0.003, while a value of 1.042 +/- 0.009 was measured for the AIB half-reaction. The secondary KIE measured for the AIB decarboxylation half-reaction with [C4'-(2)H]PLP is 0.92 +/- 0.02. The primary [2-(2)H]-L-alanine KIE on the transamination half-reaction is unity. Small but significant solvent KIEs are observed on k(cat) and k(cat)/K(M) for both substrates, and the proton inventories are linear in each case. NMR measurements of C2--H washout vs product formation give ratios of 105 and 14 with L-alanine and isopropylamine as substrates, respectively. These results support a rate-limiting, concerted C alpha-decarboxylation/C4'-protonation mechanism for the AIB decarboxylation reaction, and rapid equilibrium quinonoid formation followed by rate-limiting protonation to the ketimine intermediate for the L-alanine transamination half-reaction. Energy profiles for the two half-reactions are constructed.     

The rates of defined changes in protein structure during the catalytic cycle of lactate dehydrogenase

Rapid mixing, kinetic experiments were performed on native and modified [Tyr(3NO2)237)] porcine H4 lactate dehydrogenase at low temperatures in a medium containing 30% dimethyl sulphoxide. In the temperature range -16 to +8 degrees C, the modified enzyme-NADH complex, when mixed with 1 mM pyruvate, is converted to enzyme, NAD+ and lactate at two distinctly different rates. At -16 degrees C the more rapid process occurs at a rate of 40 s-1 and the slower at 3 s-1. The slower rate is identical to that assigned to the steady-state turnover of the enzyme in these conditions and therefore reflects the slow, rate-limiting rearrangement of protein structure which has been inferred from previous kinetic experiments. The fast phase of NADH oxidation, however, proceeds at a rate which coincides with that of the closure of a loop of polypeptide over the active site of the enzyme (sensed by the nitrotyrosine group, which protonates in response to the approach of glutamate 107, a residue situated on this mobile loop). We explain these results by proposing that: (i) both the slow and fast changes in protein structure must occur before the enzyme can accomplish the redox step, (ii) the enzyme-NADH (binary) complex exists in two, slowly interconverting forms, (iii) the structural change giving rise to this slow conformational equilibrium can also occur in the ternary (enzyme-NADH-pyruvate) complex and (iv) it is this step which limits the rate of the steady-state reaction. Both of the binary forms are able to bind pyruvate, but the rate of NADH oxidation in one of the forms is rapid, since it has already undergone this slow rearrangement. In this rapidly reacting form, it is the closure of the loop (not transfer of the hydride ion) which limits the rate at which the coenzyme is oxidized, while the slowly reacting form must undergo both loop-closure and the slow structural conversion before the redox reaction can occur.     

Catalytic role of the metal ion of carboxypeptidase A in ester hydrolysis.

The mechanism of action of bovine pancreatic carboxypeptidase. Aalpha (peptidyl-L-amino acid hydrolase; EC 3.4.12.2) has been investigated by application of cryoenzymologic methods. Kinetic studies of the hydrolysis of the specific ester substrate O-(trans-p-chlorocinnamoyl)-L-beta-phenyllactate have been carried out with both the native and the Co2+-substituted enzyme in the 25 to --45 degrees C temperature range. In the --25 to --45 degrees C temperature range with enzyme in excess, a biphasic reaction is observed for substrate hydrolysis characterized by rate constants for the fast (kf) and the slow (ks) processes. In Arrhenius plots, ks extrapolates to kcat at 25 degrees C for both enzymes in aqueous solution, indicating that the same catalytic rate-limiting step is observed. The slow process is analyzed for both metal enzymes, as previously reported (Makinen, M. W., Yamamura, K., and Kaiser, E. T. (1976) Proc Natl. Acad. Sci. U. S. A. 73, 3882-3886), to involve the deacylation of a mixed anhydride acyl-enzyme intermediate. Near --60 degrees C the acyl-enzyme intermediate of both metal enzymes can be stabilized for spectral characterization. The pH and temperature dependence of ks reveals a catalytic ionizing group with a metal ion-dependent shift in pKa and an enthalpy of ionization of 7.2 kcal/mol for the native enzyme and 6.2 kcal/mol for the Co2+ enzyme. These parameters identify the ionizing catalytic group as the metal-bound water molecule. Extrapolation of the pKa data to 25 degrees C indicates that this ionization coincides with that observed in the acidic limb of the pH profile of log(kcat/Km(app)) for substrate hydrolysis under steady state conditions. The results indicate that in the esterolytic reaction of carboxypeptidase. A deacylation of the mixed anhydride intermediate is catalyzed by a metal-bound hydroxide group.     

Structure and mechanism of a cysteine sulfinate desulfinase engineered on the aspartate aminotransferase scaffold

The joint substitution of three active-site residues in Escherichia coli (L)-aspartate aminotransferase increases the ratio of l-cysteine sulfinate desulfinase to transaminase activity 10(5)-fold. This change in reaction specificity results from combining a tyrosine-shift double mutation (Y214Q/R280Y) with a non-conservative substitution of a substrate-binding residue (I33Q). Tyr214 hydrogen bonds with O3 of the cofactor and is close to Arg374 which binds the α-carboxylate group of the substrate; Arg280 interacts with the distal carboxylate group of the substrate; and Ile33 is part of the hydrophobic patch near the entrance to the active site, presumably participating in the domain closure essential for the transamination reaction. In the triple-mutant enzyme, k(cat)' for desulfination of l-cysteine sulfinate increased to 0.5s(-1) (from 0.05s(-1) in wild-type enzyme), whereas k(cat)' for transamination of the same substrate was reduced from 510s(-1) to 0.05s(-1). Similarly, k(cat)' for β-decarboxylation of l-aspartate increased from<0.0001s(-1) to 0.07s(-1), whereas k(cat)' for transamination was reduced from 530s(-1) to 0.13s(-1). l-Aspartate aminotransferase had thus been converted into an l-cysteine sulfinate desulfinase that catalyzes transamination and l-aspartate β-decarboxylation as side reactions. The X-ray structures of the engineered l-cysteine sulfinate desulfinase in its pyridoxal-5'-phosphate and pyridoxamine-5'-phosphate form or liganded with a covalent coenzyme-substrate adduct identified the subtle structural changes that suffice for generating desulfinase activity and concomitantly abolishing transaminase activity toward dicarboxylic amino acids. Apparently, the triple mutation impairs the domain closure thus favoring reprotonation of alternative acceptor sites in coenzyme-substrate intermediates by bulk water.     

Hydrolysis of aryl beta-D-glucopyranosides and beta-D-xylopyranosides by an induced beta-D-glucosidase from Stachybotrys atra.

The induced beta-D-glucosidase from Stachybotrys atra hydrolyzes aryl beta-D-glucopyranosides and aryl beta-D-xylopyranosides by the same basic two-step mechanism. In the first step the aglycon group is split of with simultaneous formation of an enzyme-glycosyl complex. In the second step this intermediate complex reacts with water yeilding beta-D-glucose or beta-D-xylose. For beta-D-xyloside hydrolysis each of the two steps is partially rate-controlling, whereas for beta-D-glucoside hydrolysis the second step is rate-limiting. The enzyme is inhibited by high concentrations of substrate and the exact rate-concentration equation is a second-order equation. 1-Thio-beta-D-glycopyranosides with an aromatic aglycon inhibit the reaction in both a competitive and non-competitive way. A tentative mechanism is proposed to explain all types of inhibition. In this mechanism substrates and inhibitors with an aromatic aglycon group bind through hydrophobic forces to the 'aglycon subsite' of the intermediate enzyme-glycosyl complex. Binding of the second substrate molecule or of the inhibitor to this complex does not prevent the reaction of the glycosyl moiety with water, it only decreases the rate of the second step.     

The enantiomeric error frequency of aspartate aminotransferase

The enantiomeric error frequency of aspartate aminotransferase (mitochondrial isoenzyme from chicken) was assessed by adding the enzyme in high concentration (0.89 mM) to a mixture of L-glutamate and 2-oxoglutarate (12 and 1.2 mM, respectively, at pH 7.5 and 25 degrees C). The substrates continuously undergo the transamination cycle under these conditions. Thereby, L-glutamate is progressively racemized, a 1:1 ratio of two enantiomers being reached within 240 h. The enantiomeric error frequency, i.e. the ratio of the rate of D-glutamate production and the rate of the transamination reaction with glutamate and 2-oxoglutarate as substrates, is 1.5 x 10(-7). D-Glutamate is also converted to a 1:1 racemic mixture. The racemizing activity of a mixture of free pyridoxal 5'-phosphate and pyridoxamine 5'-phosphate is about two orders of magnitude lower than that of aspartate aminotransferase. The error frequency of the enzyme in the case of the C4 substrate pair aspartate and oxalacetate is 3.4 x 10(-8), i.e. 4 times lower than that with the C5 substrate pair.     

The kinetic mechanism of beef kidney D-aspartate oxidase

The mechanism of action of the flavoprotein D-aspartate oxidase (EC 1.4.3.1) has been investigated by steady-state and stopped flow kinetic studies using D-aspartate and O2 as substrates in 50 mM KPi, 0.3 mM EDTA, pH 7.4, 4 degrees C. Steady-state results indicate that a ternary complex containing enzyme, O2, and substrate (or product) is an obligatory intermediate in catalysis. The kinetic parameters are turnover number = 11.1 s-1, Km(D-Asp) = 2.2 x 10(-3) M, Km(O2) = 1.7 x 10(-4) M. Rapid reaction studies show that 1) the reductive half reaction is essentially irreversible with a maximum rate of reduction of 180 s-1; 2) the free reduced enzyme cannot be the species which is reoxidized during turnover since its reoxidation by oxygen (second order rate constant equal to 5.3 x 10(2) M-1 s-1) is too slow to be of relevance in catalysis; 3) reduced enzyme can bind a ligand rapidly and be reoxidized as a complex at a rate faster than that observed for the free reduced enzyme; 4) the rate of reoxidation of reduced enzyme by oxygen during turnover is dependent on both O2 and D-aspartate concentrations (second order rate constant of reaction between O2 and reduced enzyme-substrate complex equal to 6.2 x 10(4) M-1 s-1); and 5) the rate-limiting step in catalysis occurs after reoxidation of the enzyme and before its reduction in the following turnover. A mechanism involving reduction of enzyme by substrate, dissociation of product from reduced enzyme, binding of a second molecule of substrate to the reduced enzyme, and reoxidation of the reduced enzyme-substrate complex is proposed for the enzyme-catalyzed oxidation of D-aspartate.     

Altering the reaction specificity of eukaryotic ornithine decarboxylase

Ornithine decarboxylase (ODC) catalyzes the first committed step in the biosynthesis of polyamines, and it has been identified as a drug target for the treatment of African sleeping sickness, caused by Trypanosoma brucei. ODC is a pyridoxal 5'-phosphate (PLP) dependent enzyme and an obligate homodimer. X-ray structural analysis of the complex of the T. brucei wild-type enzyme with the product putrescine reveals two structural changes that occur upon ligand binding: Lys-69 is displaced by putrescine and forms new interactions with Glu-94 and Asp-88, and the side chain of Cys-360 rotates into the active site to within 3.4 A of the imine bond. Mutation of Cys-360 to Ala or Ser reduces the k(cat) of the decarboxylation reaction by 50- and 1000-fold, respectively. However, HPLC analysis of the products demonstrates that the mutant enzymes almost exclusively catalyze a decarboxylation-dependent transamination reaction to form pyridoxamine 5-phosphate (PMP) and gamma-aminobutyraldehyde, instead of PLP and putrescine. This side reaction arises when the decarboxylated substrate intermediate is protonated at C4' of PLP instead of at the C(alpha) of substrate. For the reaction catalyzed by the wild-type enzyme, this side reaction occurs infrequently (<0.01% of the turnovers). Single turnover analysis and multiwavelength stopped-flow spectroscopic studies suggest that for the mutant ODCs protonation at C4' occurs either very rapidly or in a concerted reaction with decarboxylation and that the rate-limiting step in the steady-state reaction is Schiff base hydrolysis/product release. These studies demonstrate a role for Cys-360 in the control of the C(alpha) protonation step that catalyzes the formation of the physiological product putrescine. The results further provide insight into the mechanism by which this class of PLP-dependent enzymes controls reaction specificity.     

Halogenated protocatechuates as substrates for protocatechuate dioxygenase from Pseudomonas cepacia

Substrates containing electron-withdrawing groups were reacted with protocatechuate 3,4-dioxygenase and oxygen. Haloprotocatechuates (5-fluoro-, 5-chloro-, 5-bromo-, 2-chloro-, and 6-chloroprotocatechuates) are oxygenated by the enzyme at rates 28- to 3000-fold lower than that with the native substrate. These lower rates are due to both deactivation of substrate to O2 attack, and to the formation of abortive enzyme-substrate (ES) complexes. Such ES complexes with haloprotocatechuates are spectrally distinct from the normal ES complex. 6-Chloroprotocatechuate produces changes more like those due to protocatechuate. The abortive ES complexes, when rapidly mixed with oxygen, decay to free enzyme and product monophasically, and the dependence of the rates on O2 concentration shows that a rate-limiting step precedes reaction with O2. Thus these complexes are rather unreactive toward O2, and the rate-limiting step in oxygenation is their conversion to active complexes. In contrast, the reaction of O2 with the enzyme and 6-chloroprotocatechuate is biphasic, the first phase being dependent on O2 concentration (2 X 10(4) M-1 S-1) and the second not (7 S-1). The intermediate formed after the first phase strongly resembles the second intermediate seen in the reaction of enzyme with protocatechuate and O2 (Bull, C., Ballou, D. P., and Otsuka, S., (1981) J. Biol. Chem. 256, 12681-12686), implying that the electron-withdrawing effect of the chlorine slows the O2 addition step considerably while the conversion to the second intermediate is hardly affected. When the enzyme cycles through several turnovers with 6-chloroprotocatechuate, an enzyme species is formed that resembles the unreactive ES complexes seen with the other haloprotocatechuates, indicating that a small amount of the unreactive complex is in equilibrium with the reactive complex and that during successive turnovers the enzyme is slowly converted into the unreactive form. The formation of this form correlates with the observation that in assays the rate of product formation gradually decreases with time.     

Kinetic analysis of the cyclin-dependent kinase-activating kinase (Cak1p) from budding yeast

Cak1p, the Cyclin-dependent kinase-activating kinase from budding yeast, is an unusual protein kinase that lacks many of the highly conserved motifs observed among members of the protein kinase superfamily. Cak1p phosphorylates and activates Cdc28p, the major cyclin-dependent kinase (CDK) in yeast, and is thereby required for passage through the yeast cell cycle. In this paper, we explore the kinetics of CDK phosphorylation by Cak1p, and we examine the role of the catalytic step in the reaction mechanism. Cak1p proceeds by a sequential reaction mechanism, binding to both ATP and CDK2 with reasonable affinities, exhibiting K(d) values of 7.2 and 0.6 microm, respectively. Interestingly, these values are approximately the same as the K(M) values, indicating that the binding of substrates is fast with respect to catalysis and that the most likely reaction mechanism is rapid equilibrium random. Cak1p is a slow enzyme, with a catalytic rate of only 4.3 min(-)(1). The absence of a burst phase indicates that product release is not rate-limiting. This result, and a solvent isotope effect, suggests that a catalytic step is rate-limiting.     

Human immunodeficiency virus-1 protease. 2. Use of pH rate studies and solvent kinetic isotope effects to elucidate details of chemical mechanism

The pH dependence of the peptidolytic reaction of recombinant human immunodeficiency virus type 1 protease has been examined over a pH range of 3-7 for four oligopeptide substrates and two competitive inhibitors. The pK values obtained from the pKis vs pH profiles for the unprotonated and protonated active-site aspartyl groups, Asp-25 and Asp-25', in the monoprotonated enzyme form were 3.1 and 5.2, respectively. Profiles of log V/K vs pH for all four substrates were "bell-shaped" in which the pK values for the unprotonated and protonated aspartyl residues were 3.4-3.7 and 5.5-6.5, respectively. Profiles of log V vs pH for these substrates were "wave-shaped" in which V was shifted to a constant lower value upon protonation of a residue of pK = 4.2-5.2. These results indicate that substrates bind only to a form of HIV-1 protease in which one of the two catalytic aspartyl residues is protonated. Solvent kinetic isotope effects were measured over a pH (D) range of 3-7 for two oligopeptide substrates, Ac-Arg-Ala-Ser-Gln-Asn-Tyr-Pro-Val-Val-NH2 and Ac-Ser-Gln-Asn-Tyr-Pro-Val-Val-NH2. The pH-independent value for DV/K was 1.0 for both substrates, and DV = 1.5-1.7 and 2.2-3.2 at low and high pH (D), respectively. The attentuation of both V and DV at low pH (D) is consistent with a change in rate-limiting step from a chemical one at high pH (D) to one in which a product release step or an enzyme isomerization step becomes partly rate-limiting at low pH (D). Proton inventory data is in accord with the concerted transfer of two protons in the transition state of a rate-limiting chemical step in which the enzyme-bound amide hydrate adduct collapses to form the carboxylic acid and amine products.     

Kinetic analysis of the mechanism of Escherichia coli dihydrofolate reductase

A kinetic mechanism is presented for Escherichia coli dihydrofolate reductase which describes the full time course of the enzymatic reaction over a wide range of substrate and enzyme concentrations at pH 7.2 and 20 degrees C. Specific rate constants were estimated by computer simulation of the full time course of single turnover, burst, and steady-state experiments using both nondeuterated and deuterated NADPH. The mechanism involves the random addition of substrates, but the substrates and enzyme are not at equilibrium prior to the chemical transformation step. The rate-limiting step follows the chemical transformation, and the maximum velocity of the reaction is limited by the release of the product tetrahydrofolate. The full time course of the reaction is markedly affected by the formation of the enzyme-NADPH-tetrahydrofolate abortive complex, but not by the enzyme-NADP-dihydrofolate abortive complex.     

Desulfuration of l-cysteine through transamination and transsulfuration in rat liver

Desulfuration of L-cysteine by rat liver via a pathway of transamination followed by transsulfuration was studied using cyanide as a sulfur acceptor. More than a 5-fold increase in formation of thiocyanate from L-cysteine and cyanide was observed in the presence of 2-oxoglutarate and pyruvate. L-Cysteine aminotransferase and 3-mercaptopyruvate sulfurtransferase activities in the same preparations were also determined. It was concluded that L-cysteine was desulfurated through transamination and transsulfuration of the resulting 3-mercaptopyruvate, and that the rate-limiting step appears to be the transamination reaction.     

Deuterium isotope effect in the transamination of p-tyrosine by rat liver tyrosine transaminase

The rate of transamination of p-tyrosine catalyzed by rat liver soluble tyrosine aminotransferase (E.C. 2.6.1.5.) was significantly reduced when the hydrogen at the alpha-carbon position is replaced by deuterium or when the reactions were conducted in 2H2O. The cleavage of carbon-hydrogen bond at alpha-carbon position is at least partly involved in the rate-limiting step of tyrosine transamination. In 2H2O solvent the reduction of the overall rates of transamination of both p-tyrosine and alpha-2H1-p-tyrosine occurred uncompetitively which suggests that the deuterium solvent effect is involved in the tautomerization of the external Schiff's base.     

Replacement of an interdomain residue Val39 of Escherichia coli aspartate aminotransferase affects the catalytic competence without altering the substrate specificity of the enzyme

Three mutant Escherichia coli aspartate aminotransferases in which Val39 was changed to Ala, Leu, and Phe by site-directed mutagenesis were prepared and characterized. Among the three mutant and the wild-type enzymes, the Leu39 enzyme had the lowest Km values for dicarboxylic substrates. The Km values of the Ala39 enzyme for dicarboxylates were essentially the same as those of the wild-type (Val39) enzyme. These two mutant enzymes showed essentially the same kcat values for dicarboxylic substrates as did the wild-type enzyme. On the other hand, incorporation of a bulky side-chain at position 39 (Phe39 enzyme) decreased both the affinity (1/Km) and catalytic ability (kcat) toward dicarboxylic substrates. These results show that the position 39 residue is involved in the modulation of both the binding of dicarboxylic substrates to enzyme and the catalytic ability of the enzyme. Although the replacement of Val39 with other residues altered both the kcat and Km values toward various substrates including dicarboxylic and aromatic amino acids and the corresponding oxo acids, it did not alter the ratio of the kcat/Km value of the enzyme toward a dicarboxylic substrate to that for an aromatic substrate. The affinity for aromatic substrates was not affected by changing the residue at position 39. These data indicate that, although the side chain bulkiness of the residue at position 39 correlates well with the activity toward aromatic substrates in the sequence alignment of several aminotransferases [Seville, M., Vincent M.G., & Hahn, K. (1988) Biochemistry 27, 8344-8349], the residue does not seem to be involved in the recognition of aromatic substrates.