General Base Catalyst Research Articles

The Role of α-Amino Group of the N-terminal Serine of β Subunit for Enzyme Catalysis and Autoproteolytic Activation of Glutaryl 7-Aminocephalosporanic Acid Acylase

Glutaryl 7-aminocephalosporanic acid (GL-7-ACA) acylase of Pseudomonas sp. strain GK16 catalyzes the cleavage of the amide bond in the GL-7-ACA side chain to produce glutaric acid and 7-aminocephalosporanic acid (7-ACA). The active enzyme is an (alphabeta)(2) heterotetramer of two non-identical subunits that are cleaved autoproteolytically from an enzymatically inactive precursor polypeptide. In this study, we prepared and characterized a chemically modified enzyme, and also examined an effect of the modification on enzyme catalysis and autocatalytic processing of the enzyme precursor. We found that treatment of the enzyme with cyanate ion led to a significant loss of the enzyme activity. Structural and functional analyses of the modified enzyme showed that carbamylation of the free alpha-amino group of the N-terminal Ser-199 of the beta subunit resulted in the loss of the enzyme activity. The pH dependence of the kinetic parameters indicates that a single ionizing group is involved in enzyme catalysis with pK(a) = 6.0, which could be attributed to the alpha-amino group of the N-terminal Ser-199. The carbamylation also inhibited the secondary processing of the enzyme precursor, suggesting a possible role of the alpha-amino group for the reaction. Mutagenesis of the invariant N-terminal residue Ser-199 confirmed the key function of its side chain hydroxyl group in both enzyme catalysis and autoproteolytic activation. Partial activity and correct processing of a mutant S199T were in agreement with the general mechanism of N-terminal nucleophile hydrolases. Our results indicate that GL-7-ACA acylase utilizes as a nucleophile Ser-199 in both enzyme activity and autocatalytic processing and most importantly its own alpha-amino group of the Ser-199 as a general base catalyst for the activation of the hydroxyl group both in enzyme catalysis and in the secondary cleavage of the enzyme precursor. All of the data also imply that GL-7-ACA acylase is a member of a novel class of N-terminal nucleophile hydrolases that have a single catalytic center for enzyme catalysis.

Kinetic mechanism of human histone acetyltransferase P/CAF.

Human transcriptional coactivator P/CAF (p300/CBP-associating factor) is a histone acetyltransferase (HAT) and is a member of the GNAT (GCN5 related N-acetyltransferases) superfamily. P/CAF was originally identified by its ability to activate transcription of a variety of genes through its interaction with p300/CBP. Though Lys-14 of histone H3 appears to be the preferred substrate, other nonhistone proteins can also serve as substrates for P/CAF. However, few studies have addressed the catalytic and kinetic mechanisms of histone/protein acetylation by P/CAF. In this study, we have systematically determined the kinetic mechanism for P/CAF, identified the critical ionizations for binding/catalysis, and established the rate-limiting step in turnover. This was accomplished by a variety of approaches including pH-dependent activity measurements, Bi-substrate kinetic analysis, authentic product inhibition by coenzyme A (CoA) and acetylated H3 (Ac-Lys-14) peptide, direct measurements of substrate/product binding affinities (equilibrium dialysis), and a pre-steady-state quench-flow analysis. The results are consistent with a fully ordered Bi-Bi kinetic mechanism, where chemical catalysis is rate-determining. Acetyl-CoA (AcCoA) binds with high affinity (K(d) = 0.64 +/- 0.12 microM) to the free form of the enzyme. Histone H3 peptide binds (apparent K(d) = 116 +/- 17 microM) only after AcCoA is bound. No H3 peptide binding to the free enzyme was detectable. In the ternary complex, the epsilon-amino of Lys-14 (H3 peptide substrate) directly attacks the carbonyl carbon of AcCoA, transferring the acetyl group to the acceptor peptide substrate (rate-limiting step). Products are released in an ordered fashion, with Ac-Lys-14 H3 released first followed by release of CoA. The pH dependency of the k(cat)/K(m) parameter revealed two P/CAF ionizable groups (pK(a) values of 6.9 and 7.5) that must be unprotonated for activity. The group with a pK(a) value 7.5 was assigned to Glu-570, which is the proposed general base catalyst, abstracting a proton from the epsilon-amino group and facilitating nucleophilic attack.

Mechanism of the phenylpyruvate tautomerase activity of macrophage migration inhibitory factor: properties of the P1G, P1A, Y95F, and N97A mutants.

Phenylpyruvate tautomerase (PPT) has been studied periodically since its activity was first described over forty years ago. In the last two years, the mechanism of PPT has been investigated more extensively because of the discovery that PPT is the same protein as the immunoregulatory cytokine known as macrophage migration inhibitory factor (MIF). The mechanism of PPT is likely to involve general base-general acid catalysis. While several lines of evidence implicate Pro-1 as the general base, the identity of the general acid remains unknown. Crystal structures of MIF with the competitive inhibitor (E)-2-fluoro-p-hydroxycinnamate bound in the active site and that of the protein complexed with the enol form of a substrate, (p-hydroxyphenyl)pyruvate, suggest that Tyr-95 is the only candidate in the vicinity that can function as a general acid catalyst. Although Tyr-95 is nearby the bound inhibitor and substrate, it is not within hydrogen bonding distance of either ligand. In this study, Tyr-95 was mutated to phenylalanine, and the kinetic and structural properties of the Y95F mutant were determined. This alteration produces a fully active enzyme, which shows no significant structural changes in the active site. The results indicate that Tyr-95 does not function as the general acid catalyst in the reaction catalyzed by wild-type PPT. The mechanism of PPT was studied further by constructing and characterizing the kinetic properties of two mutants of Pro-1 (P1G and P1A) and one mutant of Asn-97 (N97A). The mutation of Asn-97, a residue implicated in the binding of the phenolic hydroxy group of the keto and enol isomers of (p-hydroxyphenyl)pyruvate and of (E)-2-fluoro-p-hydroxycinnamate affects only the binding affinity of the inhibitor. However, the mutations of Pro-1 have a profound effect on the values of k(cat) and k(cat)/K(m) and clearly show that Pro-1 is a critical residue in the reaction. The results are discussed in terms of a mechanism in which Pro-1 functions as both the general acid and the general base catalyst.

Ca2+-assisted, direct hydride transfer, and rate-determining tautomerization of C5-reduced PQQ to PQQH2, in the oxidation of beta-D-glucose by soluble, quinoprotein glucose dehydrogenase.

Spectral and kinetic studies were performed on enzyme forms of soluble glucose dehydrogenase of the bacterium Acinetobacter calcoaceticus (sGDH) in which the PQQ-activating Ca(2+) was absent (Holo X) or was replaced with Ba(2+) (Ba-E) or in which PQQ was replaced with an analogue or a derivative called "nitroPQQ" (E-NPQ). Although exhibiting diminished rates, just like sGDH, all enzyme forms were able to oxidize a broad spectrum of aldose sugars, and their reduced forms could be oxidized with the usual artificial electron acceptor. On inspection of the plots for the reductive half-reaction, it appeared that the enzyme forms exhibited a negative cooperativity effect similar to that of sGDH itself under turnover conditions, supporting the view that simultaneous binding of substrate to the two subunits of sGDH causes the effect. Stopped-flow spectroscopy of the reductive half-reaction of Ba-E with glucose showed a fluorescing transient previously observed in the reaction of sGDH with glucose-1-d, whereas no intermediate was detected at all in the reactions of E-NPQ and Holo X. Using hydrazine as a probe, the fluorescing C5 adduct of PQQ and hydrazine was formed in sGDH, Ba-E, and Holo X, but E-NPQ did not react with hydrazine. When this is combined with other properties of E-NPQ and the behavior of enzyme forms containing a PQQ analogue, we concluded that the catalytic potential of the cofactor in the enzyme is not determined by its adduct-forming ability but by whether it is or can be activated with Ca(2+), activation being reflected by the large red shift of the absorption maximum induced by this metal ion when binding to the reduced cofactor in the enzyme. This conclusion, together with the observed deuterium kinetic isotope effect of 7.8 on transient formation in Ba-E, and that already known on transient decay, indicate that the sequential steps in the mechanism of sGDH must be (1) reversible substrate binding, (2) direct transfer of a hydride ion (reversible or irreversible) from the C1 position of the beta-anomer of glucose to the C5 of PQQ, (3) irreversible, rate-determining tautomerization of the fluorescing, C5-reduced PQQ to PQQH(2) and release (or earlier) of the product, D-glucono-delta-lactone, and (4) oxidation of PQQH(2) by an electron acceptor. The PQQ-activating Ca(2+) greatly facilitates the reactions occurring in step 2. His144 may also play a role in this by acting as a general base catalyst, initiating hydride transfer by abstracting a proton from the anomeric OH group of glucose. The validity of the proposed mechanism is discussed for other PQQ-containing dehydrogenases.

Identification of key amino acid residues in Neisseria polysaccharea amylosucrase

Amylosucrase from Neisseria polysaccharea catalyzes the synthesis of an amylose-like polymer from sucrose. Sequence alignment revealed that it belongs to the glycoside hydrolase family 13. Site-directed mutagenesis enabled the identification of functionally important amino acid residues located at the active center. Asp-294 is proposed to act as the catalytic nucleophile and Glu-336 as general acid base catalyst in amylosucrase. The conserved Asp-401, His-195 and His-400 residues are critical for the enzymatic activity. These results provide strong support for the predicted close structural and functional relationship between the sucrose-glucosyltransferases and enzymes of the α-amylase family.

Kinetics of Two Pathways in Peroxyoxalate Chemiluminescence

It has been shown that 1,1'-oxalyldiimidazole (ODI) is formed as an intermediate in the imidazole-catalyzed reaction of oxalate esters with hydrogen peroxide. Therefore, the kinetics of the chemiluminescence reaction of 1,1'-oxalyldiimidazole (ODI) with hydrogen peroxide in the presence of a fluorophore was investigated in order to further elucidate the mechanism of the peroxyoxalate chemiluminescence reaction. The effects of concentrations of ODI, hydrogen peroxide, imidazole (ImH), the general-base catalysts lutidine and collidine, and temperature on the chemiluminescence profile and relative quantum efficiency in the solvent acetonitrile were determined using the stopped-flow technique. Pseudo-first-order rate constant measurements were made for concentrations of either H2O2 or ODI in large excess. All of the reaction kinetics are consistent with a mechanism in which the reaction is initiated by a base-catalyzed substitution of hydrogen peroxide for imidazole in ODI to form an imidazoyl peracid (Im(CO)2OOH). In the presence of a large excess of H2O2, this intermediate rapidly decays with both a zero- and first-order dependence on the H2O2 concentration. It is proposed that the zero-order process reflects a cyclization of this intermediate to form a species capable of exciting a fluorophore via the "chemically initiated electron exchange mechanism" (CIEEL), while the first-order process results from the substitution of an additional molecule of hydrogen peroxide to the imidazoyl peracid to form dihydroperoxyoxalate, reducing the observed quantum yield. Under conditions of a large excess of ODI, the reaction is more than 1 order of magnitude more efficient at producing light, and the quantum yield increases linearly with increasing ODI concentration. Again, it is proposed that the slow initiating step of the reaction involves the substitution of H2O2 for imidazole to form the imidazoyl peracid. This intermediate may decay by either cyclization or by reaction with another ODI molecule to form a cyclic peroxide that is much more efficient at energy transfer with the fluorophore. The reaction kinetics clearly distinguishes two separate pathways for the chemiluminescent reaction.

Lipases provide a new mechanistic model for polyhydroxybutyrate (PHB) synthases: characterization of the functional residues in Chromatium vinosum PHB synthase.

Polyhydroxybutyrate (PHB) synthases catalyze the conversion of beta-hydroxybutyryl coenzyme A (HBCoA) to PHB. These enzymes require an active site cysteine nucleophile for covalent catalysis. A protein BLASTp search using the Class III Chromatium vinosum synthase sequence reveals high homology to prokaryotic lipases whose crystal structures are known. The homology is very convincing in the alpha-beta-elbow (with the active site nucleophile)-alpha-beta structure, residues 131-175 of the synthase. A conserved histidine of the Class III PHB synthases aligns with the active site histidine of the lipases using the ClustalW algorithm. This is intriguing as this histidine is approximately 200 amino acids removed in sequence space from the catalytic nucleophile. Different threading algorithms suggest that the Class III synthases belong to the alpha/beta hydrolase superfamily which includes prokaryotic lipases. Mutagenesis studies were carried out on C. vinosum synthase C149, H331, H303, D302, and C130 residues. These studies reveal that H331 is the general base catalyst that activates the nucleophile, C149, for covalent catalysis. The model indicates that C130 is not involved in catalysis as previously proposed [Müh, U., Sinskey, A. J., Kirby, D. P., Lane, W. S., and Stubbe, J. (1999) Biochemistry 38, 826-837]. Studies with D302 mutants suggest D302 functions as a general base catalyst in activation of the 3-hydroxyl of HBCoA (or a hydroxybutyrate acyl enzyme) for nucleophilic attack on the covalently linked thiol ester intermediate. The relationship of the lipase model to previous models based on fatty acid synthases is discussed.

Physical Organic Chemistry of Transition Metal Carbene Complexes. 18.1 Kinetics of Reversible Cyclization of the Fischer Carbene Complexes (CO)5MC(SCH2CH2OH)Ph and (CO)5MC(OCH2CH2OH)Ph (M = Cr, W) in Aqueous Acetonitrile

Rate and equilibrium constants for the conversion of (CO)5CrC(OCH2CH2OH)Ph (8-CrOOH), (CO)5WC(OCH2CH2OH)Ph (8-WOOH), (CO)5CrC(SCH2CH2OH)Ph (8-CrSOH), and (CO)5WC(SCH2CH2OH)Ph (8-WSOH) into the corresponding cyclic adducts (9-CrOO- for M = Cr, X = O; 9-WOO- for M = W, X = O; 9-CrSO- for M = Cr, X = S; 9-WSO- for M = W, X = S) are reported. Under basic conditions the reaction occurs via the respective oxyanions, (CO)5CrC(OCH2CH2O-)Ph (8-CrOO-), etc.; under acidic conditions the reaction may either be concerted with water acting as a general base catalyst or stepwise via a zwitterionic intermediate. The absence of buffer catalysis suggests but does not prove the operation of the stepwise mechanism. The equilibrium constants for cyclization of 8-CrSO- and 8-WSO- are about 1 order of magnitude higher than for 8-CrOO- and 8-WOO-, respectively. For the rate constants the situation is reversed, with 8-CrOO- and 8-WOO- being substantially more reactive than 8-CrSO- and 8-WSO-, respectively. These results reflect t...

Effects of mutations of the active site arginine residues in 4-oxalocrotonate tautomerase on the pKa values of active site residues and on the pH dependence of catalysis.

The unusually low pK(a) value of the general base catalyst Pro-1 (pK(a) = 6.4) in 4-oxalocrotonate tautomerase (4-OT) has been ascribed to both a low dielectric constant at the active site and the proximity of the cationic residues Arg-11 and Arg-39 [Stivers, J. T., Abeygunawardana, C., Mildvan, A. S., Hajipour, G., and Whitman, C. P. (1996) Biochemistry 35, 814-823]. In addition, the pH-rate profiles in that study showed an unidentified protonated group essential for catalysis with a pK(a) of 9.0. To address these issues, the pK(a) values of the active site Pro-1 and lower limit pK(a) values of arginine residues were determined by direct (15)N NMR pH titrations. The pK(a) values of Pro-1 and of the essential acid group were determined independently from pH-rate profiles of the kinetic parameters of 4-OT in arginine mutants of 4-OT and compared with those of wild type. The chemical shifts of all of the Arg Nepsilon resonances in wild-type 4-OT and in the R11A and R39Q mutants were found to be independent of pH over the range 4.9-9.7, indicating that no arginine is responsible for the kinetically determined pK(a) of 9.0 for an acidic group in free 4-OT. With the R11A mutant, where k(cat)/K(m) was reduced by a factor of 10(2.9), the pK(a) of Pro-1 was not significantly altered from that of the wild-type enzyme (pK(a) = 6.4 +/- 0.2) as revealed by both direct (15)N NMR titration (pK(a) = 6.3 +/- 0.1) and the pH dependence of k(cat)/K(m) (pK(a) = 6.4 +/- 0.2). The pH-rate profiles of both k(cat)/K(m) and k(cat) for the reaction of the R11A mutant with the dicarboxylate substrate, 2-hydroxymuconate, showed humps, i.e., sharply defined maxima followed by nonzero plateaus. The humps disappeared in the reaction with the monocarboxylate substrate, 2-hydroxy-2,4-pentadienoate, indicating that, unlike the wild-type enzyme which reacts only with the dianionic form of the dicarboxylic substrate, the R11A mutant reacts with both the 6-COOH and 6-COO(-) forms, with the 6-COOH form being 12-fold more active. This reversal in the preferred ionization state of the 6-carboxyl group of the substrate that occurs upon mutation of Arg-11 to Ala provides strong evidence that Arg-11 interacts with the 6-carboxylate of the substrate. In the R39Q mutant, where k(cat)/K(m) was reduced by a factor of 10(3), the kinetically determined pK(a) value for Pro-1 was 4.6 +/- 0.2, while the ionization of Pro-1 showed negative cooperativity with an apparent pK(a) of 7.1 +/- 0.1 determined by 1D (15)N NMR. From the Hill coefficient of 0.54, it can be shown that the apparent pK(a) value of 7.1 could result most simply from the averaging of two limiting pK(a) values of 4.6 and 8.2. Mutation of Arg-39, by altering the structure of the beta-hairpin which covers the active site, could result in an increase in the solvent exposure of Pro-1, raising its upper limit pK(a) value to 8.2. In the R39A mutant, the kinetically determined pK(a) of Pro-1 was also low, 5.0 +/- 0.2, indicating that in both the R39Q and R39A mutants, only the sites with low pK(a) values were kinetically operative. With the fully active R61A mutant, the kinetically determined pK(a) of Pro-1 (pK(a) = 6.5 +/- 0.2) agreed with that of wild-type 4-OT. It is concluded that the unusually low pK(a) of Pro-1 shows little contribution from electrostatic effects of the nearby cationic Arg-11, Arg-39, and Arg-61 residues but results primarily from a site of low local dielectric constant.

Kinetic and Molecular-Modelling Studies of Reactions of a Class-Aβ-Lactamase with Compounds Bearing a Methoxy Group on theβ-Lactam Ring

The interactions between Staphylococcus aureus PC1 enzyme and compounds bearing a methoxy group on the α-face of the β-lactam ring (cefmetazole (1), moxalactam (2) and cefoxitin (3)) were studied. With these compounds, a partitioning of the acyl-enzyme between deacylation and a transiently inactivated form of the enzyme were observed. The individual microscopic rate constants were determined, indicating for 1 and 3 that k′3>k3 (Scheme 1), whereas for 2 k′3≈k3. This different behavior could be attributed to the presence of the α-carboxy group at the C(7) side chain of 2, which is able to act as a general-base catalyst in the deacylation step. Molecular-modelling studies allowed correlation of KS and k2 with the structures of the Henri-Michaelis complexes that these compounds formed with the S. aureus enzyme. The acylation rate constant (k2) for these `β-lactamase-stable' compounds was lower than that observed with substrates lacking the methoxy group. Molecular-modelling studies indicated that the methoxy group increases the displacement of the crystallographically observed water molecule (Wat81), which is involved in the acylation mechanism. On the other hand, an average of the most important interactions in the Henri-Michaelis complexes was related to KS. An increase of 0.2 – 0.5 Å in this average value was found to result in an increase in KS by about one order of magnitude.

Lysine 183 is the general base in the 6-phosphogluconate dehydrogenase-catalyzed reaction.

Site-directed mutagenesis was used to change K183 of sheep liver 6-phosphogluconate dehydrogenase to A, E, H, C, Q, R, and M to probe its possible role as a general base catalyst. Each of the mutant proteins was characterized with respect to its kinetic parameters at pH 7 and the pH dependence of kinetic parameters for the K183R mutant enzyme. The only mutant enzyme that gives a significant amount of catalysis is the K183R mutant, and the extent of catalysis is decreased by about 3 orders of magnitude; the general base pK is perturbed to a pH value of >9. All other mutant enzymes exhibit rates that are decreased by about 4 orders of magnitude compared to that of the wild-type enzyme. Data are consistent with the general base function of K183.

Specificity and catalysis of uracil DNA glycosylase. A molecular dynamics study of reactant and product complexes with DNA.

The structure of uracil DNA glycosylase (UDG) in complex with a nonamer duplex DNA containing a uracil has been determined only in the product state. The reactant state was constructed by reattaching uracil to the deoxyribose, and both complexes were studied by molecular dynamics simulations. Significant changes in the positions of secondary structural elements in the enzyme are induced by the hydrolysis of the glycosidic bond. The simulations show that the specificity of the uracil pocket in the enzyme is largely retained in both complexes with the exception of Asn-204, which has been identified as a residue that contributes to discrimination between uracil and cytosine. The hydrogen bond between the amide group of Asn-204 and O(4) of uracil is disrupted by fluctuations of the side chain in the reactant state and is replaced by a hydrogen bond to water molecules trapped in the interior of the protein behind the uracil binding pocket. The role of two residues implicated by mutation experiments to be important in catalysis, His-268 and Asp-145, is clarified by the simulations. In the reactant state, His-268 is found 3.45 +/- 0.34 A from the uracil, allowing a water molecule to form a bridge to O(2). The environment in the enzyme raises the pK(a) value of His-268 to 7.1, establishing a protonated residue for assisting in the hydrolysis of the glycosidic bond. In agreement with the crystallographic structure, the DNA backbone retracts after the hydrolysis to allow His-268 to approach the O(2) of uracil with a concomitant release of the bridging water molecule and a reduction in the pK(a) to 5.5, which releases the proton to the product. The side chain of Asp-145 is fully solvated in the reactant state and H-bonded through a water molecule to the 3'-phosphate of uridine. Both the proximity of Asp-145 to the negatively charged phosphate and its pK(a) of 4.4 indicate that it cannot act as a general base catalyst. We propose a mechanism in which the bridging water between Asp-145 and the 3'-phosphate accepts a proton from another water to stabilize the bridge through a hydronium ion as well as to produce the hydroxide anion required for the hydrolytic step. The mechanism is consistent with known experimental data.

Catalytic Mechanism and Function of Invariant Glutamic Acid 173 from the Histone Acetyltransferase GCN5 Transcriptional Coactivator

Within chromatin, reversible acetylation of core histones is critical for transcriptional activation of eukaryotic target genes. The recent identification of intrinsic histone acetyltransferase (HAT) catalytic activity from a number of transcriptional co-activators (including yeast GCN5, p300/CBP, P/CAF, and TAFII250), has underscored the importance of protein acetylation in transcriptional control. The GCN5 family is the prototype for a diverse group of at least four distinct human HATs families. Although there is now a clear link between in vivo HAT catalytic activity and gene activation, little is known about the molecular mechanisms of histone acetylation. Herein, we report the first detailed biochemical study that probes the catalytic mechanism and the function of invariant glutamic acid 173 within the GCN5 family of HATs. Our results suggest that the HAT reaction involves the formation of a ternary complex (histones, acetyl-CoA, and enzyme) where the epsilon-amino group of histone lysine residues directly attacks the bound acetyl-CoA. The acetylation reaction requires deprotonation of the epsilon-amino group prior to nucleophilic attack. Employing site-directed mutagenesis, chemical modification, steady-state, and pH-dependent rate analysis, it is demonstrated that glutamic acid 173 is an essential catalytic residue, acting as a general base catalyst by deprotonating the histone substrate.

Crystal structure determination of cholesterol oxidase from Streptomyces and structural characterization of key active site mutants.

Cholesterol oxidase is a monomeric flavoenzyme which catalyzes the oxidation and isomerization of cholesterol to cholest-4-en-3-one. The enzyme interacts with lipid bilayers in order to bind its steroid substrate. The X-ray structure of the enzyme from Brevibacterium sterolicum revealed two loops, comprising residues 78-87 and residues 433-436, which act as a lid over the active site and facilitate binding of the substrate [Vrielink et al. (1991) J. Mol. Biol. 219, 533-554; Li et al. (1993) Biochemistry 32, 11507-11515]. It was postulated that these loops must open, forming a hydrophobic channel between the membrane and the active site of the protein and thus sequestering the cholesterol substrate from the aqueous environment. Here we describe the three-dimensional structure of the homologous enzyme from Streptomyces refined to 1.5 A resolution. Structural comparisons to the enzyme from B. sterolicum reveal significant conformational differences in these loop regions; in particular, a region of the loop comprising residues 78-87 adopts a small amphipathic helical turn with hydrophobic residues directed toward the active site cavity and hydrophilic residues directed toward the external surface of the molecule. It seems reasonable that this increased rigidity reduces the entropy loss that occurs upon binding substrate. Consequently, the Streptomyces enzyme is a more efficient catalyst. In addition, we have determined the structures of three active site mutants which have significantly reduced activity for either the oxidation (His447Asn and His447Gln) or the isomerization (Glu361Gln). Our structural and kinetic data indicate that His447 and Glu361 act as general base catalysts in association with conserved water H2O541 and Asn485. The His447, Glu361, H2O541, and Asn485 hydrogen bond network is conserved among other oxidoreductases. This catalytic tetrad appears to be a structural motif that occurs in flavoenzymes that catalyze the oxidation of unactivated alcohols.

Lysine-313 of 5-aminolevulinate synthase acts as a general base during formation of the quinonoid reaction intermediates.

5-Aminolevulinate synthase catalyzes the condensation of glycine and succinyl-CoA to form CoA, carbon dioxide, and 5-aminolevulinate. This represents the first committed step of heme biosynthesis in animals and some bacteria. Lysine 313 (K313) of mature murine erythroid 5-aminolevulinate synthase forms a Schiff base linkage to the pyridoxal 5'-phosphate cofactor. In the presence of glycine and succinyl-CoA, a quinonoid intermediate absorption is transiently observed in the visible spectrum of purified murine erythroid ALAS. Mutant enzymes with K313 replaced by glycine, histidine, or arginine exhibit no spectral evidence of quinonoid intermediate formation in the presence of glycine and succinyl-CoA. The wild-type 5-aminolevulinate synthase additionally forms a stable quinonoid intermediate in the presence of the product, 5-aminolevulinate. Only conservative mutation of K313 to histidine or arginine produces a variant that forms a quinonoid intermediate with 5-aminolevulinate. The quinonoid intermediate absorption of these mutants is markedly less than that of the wild-type enzyme, however. Whereas the wild-type enzyme catalyzes loss of tritium from [2-3H2]-glycine, mutation of K313 to glycine results in loss of this activity. Titration of the quinonoid intermediate formed upon binding of 5-aminolevulinate to the wild-type enzyme indicated that the quinonoid intermediate forms by transfer of a single proton with a pK of 8.1 +/- 0.1. Conservative mutation of K313 to histidine raises this value to 8.6 +/- 0.1. We propose that K313 acts as a general base catalyst to effect quinonoid intermediate formation during the 5-aminolevulinate synthase catalytic cycle.

Identification of a Catalytic Aspartyl Residue ofd-Ribulose 5-Phosphate 3-Epimerase by Site-directed Mutagenesis

Guided by comparative sequence considerations, we have examined the possibility of a catalytic role of Asp186 of D-ribulose 5-phosphate epimerase by site-directed mutagenesis of the recombinant spinach enzyme. Accordingly, D186A, D186N, and D186E mutants of the epimerase were constructed, purified, and characterized; as judged by their electrophoretic mobilities the mutants are properly assembled into octamers like the wild-type enzyme. Based on the extent of internal quenching of Trp fluorescence, the conformational integrity of the wild-type enzyme is preserved in the mutants. The wild-type kcat of 7.1 x 10(3) s-1 is lowered to 3.3 x 10(-4) s-1 in D186A, 0.13 s-1 in D186N, and 1.1 s-1 in D186E; as gauged by D186A, altogether lacking a functional side chain at position 186, the beta-carboxyl of Asp186 facilitates catalysis by >10(7)-fold. Relative to the wild-type enzyme, the Km for D-ribulose 5-phosphate is essentially unaltered with D186N and D186E but increased 10-fold with D186A. Apart from their impairments in epimerase activity, the mutants are unable to catalyze exchange between solvent protons and the C3 proton of substrates. This deficiency and the differential alterations of kinetic parameters among the mutants are consistent with Asp186 serving as an electrophile to facilitate alpha-proton abstraction. This study is the first to identify a catalytic group of the epimerase.

Tyrosine 146 of thymidylate synthase assists proton abstraction from the 5-position of 2'-deoxyuridine 5'-monophosphate.

Tyr 146 of TS has been proposed to assist in the removal of the proton from the 5-carbon of the pyrimidine in a steady-state intermediate [Hyatt, D. C., Maley, F., and Montfort, W. R. (1997) Biochemistry 36, 4585-4594]. We prepared a replacement set of mutations at position 146 of L. casei TS. The kcat and kcat/Km values of 15 mutants studied were significantly lower than wild-type TS. There was no effect on the Km of dUMP, and only moderate effects on the Km of the cofactor. We concluded that Y146 is not directly involved in substrate binding, but contributes significantly to catalysis. We also examined the Y146 mutants as catalysts for cofactor-independent dehalogenation of BrdUMP, a reaction which simulates early steps of the normal pathway up to and including enzyme-nucleotide covalent adduct formation. Many mutants had activity comparable to the wild-type enzyme, and we concluded that the effects of Tyr 146 mutations occur after the initial covalent adduct is formed. A covalent steady-state intermediate-containing enzyme, dUMP, and cofactor accumulated with Tyr 146 mutants, and could be isolated by SDS-PAGE. The complex was kinetically competent as an intermediate in dTMP formation. Using Y146D and F, it was shown that removal of the C-5 proton from the covalent intermediate was defective. We conclude that in the wild-type enzyme Tyr 146 assists in proton removal from the covalent intermediate. Mutants containing fluorinated tyrosines at position 146 showed an inverse linear correlation of activity versus acidity, again indicating that the basicity of the phenolic oxygen plays an important catalytic role. Speculations of how the poorly basic phenol group might assist proton removal are made in which Tyr 146 acts as a proton conduit to N5 of the cofactor or as a cohort of a water molecule serving as the direct general base catalyst.

Evaluation of the role of His447 in the reaction catalyzed by cholesterol oxidase.

Cholesterol oxidase catalyzes the oxidation and isomerization of cholesterol to cholest-4-en-3-one via cholest-5-en-3-one. It has been proposed that His447 acts as the general base catalyst for oxidation, and that the resulting imidazolium ion formed acts as an electrophile for isomerization. In this work, we undertook an assessment of the proposed dual roles of His447 in the oxidation and isomerization reactions. To test its role, we constructed five mutants, H447Q, H447N, H447E, H447D, and H447K, that introduce hydrogen bond donors and acceptors and carboxylate bases at this position, and a sixth mutant, E361Q, to test the interplay between His447 and Glu361. These mutants were characterized using steady-state kinetics and deuterium substrate and solvent isotope effects. For those mutants that catalyze either oxidation of cholesterol or isomerization of cholest-5-en-3-one, the Km's vary no more than 3-fold relative to wild type. H447K is inactive in both oxidation (> 100,000-fold reduced) and isomerization assays (> 10,000-fold reduced). H447E and H447D do not catalyze oxidation (> 100,000-fold reduced), but do catalyze isomerization, 10(4) times slower than wild type. The k(cat) for H447Q is 120-fold lower than wild type for oxidation, and the same as wild type for isomerization. The k(cat) for H447N is 4400-fold lower than wild type for oxidation, and is 30-fold lower than wild type for isomerization. E361Q does not catalyze isomerization (> 10,000-fold reduced), and the k(cat) for oxidation is 30-fold lower than wild type. The substrate deuterium kinetic isotope effects for the wild-type and mutant-catalyzed oxidation reactions suggest that mutation of His447 to an amide results in a change of the rate-determining step from hydride transfer to hydroxyl deprotonation. The deuterium solvent and substrate kinetic isotope effects for isomerization indicate that an amide at position 447 is an effective electrophile to catalyze formation of a dienolic intermediate. Moreover, consideration of kinetic and structural results together suggests that a hydrogen bonding network involving His447, Glu361 and Asn485, Wat541, and substrate serves to position the substrate and coordinate general base and electrophilic catalysis. That is, in addition to its previously demonstrated role as base for deprotonation of carbon-4 during isomerization, Glu361 has a structural role and may act as a general base during oxidation. The His447, Asn485, Glu361, and Wat541 residues are conserved in other GMC oxidoreductases. Observation of this catalytic tetrad in flavoproteins of unknown function may be diagnostic for an ability to oxidize unactivated alcohols.

Characterization of the role of the amino-terminal proline in the enzymatic activity catalyzed by macrophage migration inhibitory factor.

The cytokine macrophage migration inhibitory factor (MIF) mediates several immune and inflammatory processes through unknown or poorly understood mechanisms. The protein shares structural homology with two bacterial isomerases, 4-oxalocrotonate tautomerase (4-OT) and 5-(carboxymethyl)-2-hydroxymuconate isomerase (CHMI), and catalyzes the enolization of phenylpyruvate and the ketonization of (p-hydroxyphenyl)pyruvate. The amino-terminal proline has been identified as the catalytic base in both the 4-OT- and CHMI-catalyzed reactions. MIF also has an amino-terminal proline that has been implicated as a catalytic group in the MIF-catalyzed reaction. To delineate further the role of Pro-1 in the MIF-catalyzed reaction, affinity labeling studies were performed with 3-bromopyruvate (3-BP). The results of this study show that 3-BP acts as an active-site-directed irreversible inhibitor of the enzymatic activity and modifies one site per monomeric subunit. The inhibitor, as its lactyl derivative, is covalently attached to an 11 residue amino-terminal fragment, Pro-1 to Arg-11. The only reasonable site for alkylation within this peptide fragment is the amino-terminal proline. Because the pKa measured for the pH dependence of kinact/KI (5.7 +/- 0.2) and that measured for the pH dependence of the kcat/Km for the enolization of phenylpyruvate (6.0 +/- 0.1) are comparable and in reasonable agreement with the previously measured pKa of Pro-1 (5.6 +/- 0.1) obtained by its direct titration [Swope, M., Sun H.-W., Blake, P., and Lolis, E. (1998) EMBO J. (in press)], it is concluded that Pro-1 acts as the general base catalyst in the MIF-catalyzed reaction. The structural and mechanistic parallels place 4-OT, CHMI, and MIF in a superfamily of enzymes related by their ability to catalyze the keto-enol tautomerization of a pyruvyl moiety.

An amino acid as a cofactor for a catalytic polynucleotide.

Natural ribozymes require metal ion cofactors that aid both in structural folding and in chemical catalysis. In contrast, many protein enzymes produce dramatic rate enhancements using only the chemical groups that are supplied by their constituent amino acids. This fact is widely viewed as the most important feature that makes protein a superior polymer for the construction of biological catalysts. Herein we report the in vitro selection of a catalytic DNA that uses histidine as an active component for an RNA cleavage reaction. An optimized deoxyribozyme from this selection requires L-histidine or a closely related analog to catalyze RNA phosphoester cleavage, producing a rate enhancement of approximately 1-million-fold over the rate of substrate cleavage in the absence of enzyme. Kinetic analysis indicates that a DNA-histidine complex may perform a reaction that is analogous to the first step of the proposed catalytic mechanism of RNase A, in which the imidazole group of histidine serves as a general base catalyst. Similarly, ribozymes of the "RNA world" may have used amino acids and other small organic cofactors to expand their otherwise limited catalytic potential.