Methanobacterium thermoformicicum thymine DNA mismatch glycosylase: conversion of an N-glycosylase to an AP lyase.

The thymine DNA mismatch glycosylase from Methanobacterium thermoformicicum, a member of the endonuclease III family of repair proteins, excises the pyrimidine base from T-G and U-G mismatches. Unlike endonuclease III, it does not cleave the phosphodiester backbone by a beta-elimination reaction. This cleavage event has been attributed to a nucleophilic attack by the conserved Lys120 of endonuclease III on the aldehyde group at C1' of the deoxyribose and subsequent Schiff base formation. The inability of TDG to perform this beta-elimination event appears to be due to the presence of a tyrosine residue at the position equivalent to Lys120 in endonuclease III. The purpose of this work was to investigate the requirements for AP lyase activity. We replaced Tyr126 in TDG with a lysine residue to determine if this replacement would yield an enzyme with an associated AP lyase activity capable of removing a mismatched pyrimidine. We observed that this replacement abolishes the glycosylase activity of TDG but does not affect substrate recognition. It does, however, convert the enzyme into an AP lyase. Chemical trapping assays show that this cleavage proceeds through a Schiff base intermediate and suggest that the amino acid at position 126 interacts with C1' on the deoxyribose sugar.     

Role of the N-terminal proline residue in the catalytic activities of the Escherichia coli Fpg protein

The Escherichia coli Fpg protein is a DNA glycosylase/AP lyase. It removes, in DNA, oxidized purine residues, including the highly mutagenic C8-oxo-guanine (8-oxoG). The catalytic mechanism is believed to involve the formation of a transient Schiff base intermediate formed between DNA containing an oxidized residue and the N-terminal proline of the Fpg protein. The importance and the role of this proline upon the various catalytic activities of the Fpg protein was examined by targeted mutagenesis, resulting in the construction of three mutant Fpg proteins: Pro-2 --> Gly (FpgP2G), Pro-2 --> Thr (FpgP2T), and Pro-2 --> Glu (FpgP2E). The formamidopyrimidine DNA glycosylase activities of FpgP2G and FpgP2T were comparable and accounted for 10% of the wild-type activity. FpgP2G and FpgP2T had barely detectable 8-oxoG-DNA glycosylase activity and produced minute Schiff base complex with 8-oxoG/C DNA. FpgP2G and FpgP2T mutants did not cleave a DNA containing preformed AP site but readily produced Schiff base complex with this substrate. FpgP2E was completely inactive in all the assays. The binding constants of the different mutants when challenged with a duplex DNA containing a tetrahydrofuran residue were comparable. The mutant Fpg proteins barely or did not complement in vivo the spontaneous transitions G/C --> T/A in E. coli BH990 (fpg mutY) cells. These results show the mandatory role of N-terminal proline in the 8-oxoG-DNA glycosylase activity of the Fpg protein in vitro and in vivo as well as in its AP lyase activity upon preformed AP site but less in the 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine-DNA glycosylase activity.     

High resolution reaction intermediates of rabbit muscle fructose-1,6-bisphosphate aldolase: substrate cleavage and induced fit

Crystal structures were determined to 1.8 A resolution of the glycolytic enzyme fructose-1,6-bis(phosphate) aldolase trapped in complex with its substrate and a competitive inhibitor, mannitol-1,6-bis(phosphate). The enzyme substrate complex corresponded to the postulated Schiff base intermediate and has reaction geometry consistent with incipient C3-C4 bond cleavage catalyzed Glu-187, which is adjacent by to the Schiff base forming Lys-229. Atom arrangement about the cleaved bond in the reaction intermediate mimics a pericyclic transition state occurring in nonenzymatic aldol condensations. Lys-146 hydrogen-bonds the substrate C4 hydroxyl and assists substrate cleavage by stabilizing the developing negative charge on the C4 hydroxyl during proton abstraction. Mannitol-1,6-bis(phosphate) forms a noncovalent complex in the active site whose binding geometry mimics the covalent carbinolamine precursor. Glu-187 hydrogen-bonds the C2 hydroxyl of the inhibitor in the enzyme complex, substantiating a proton transfer role by Glu-187 in catalyzing the conversion of the carbinolamine intermediate to Schiff base. Modeling of the acyclic substrate configuration into the active site shows Glu-187, in acid form, hydrogen-bonding both substrate C2 carbonyl and C4 hydroxyl, thereby aligning the substrate ketose for nucleophilic attack by Lys-229. The multifunctional role of Glu-187 epitomizes a canonical mechanistic feature conserved in Schiff base-forming aldolases catalyzing carbohydrate metabolism. Trapping of tagatose-1,6-bis(phosphate), a diastereoisomer of fructose 1,6-bis(phosphate), displayed stereospecific discrimination and reduced ketohexose binding specificity. Each ligand induces homologous conformational changes in two adjacent alpha-helical regions that promote phosphate binding in the active site.     

Active-site-directed inactivation of wheat-germ aspartate transcarbamoylase by pyridoxal 5''-phosphate.

Treatment of 1 microM wheat-germ aspartate transcarbamoylase with 1 mM-pyridoxal 5'-phosphate caused a rapid loss of activity, concomitant with the formation of a Schiff base. Complete loss of activity occurred within 10 min when the Schiff base was reduced with a 100-fold excess of NaBH4. Concomitantly, one amino group per chain was modified. No further residues were modified in the ensuing 30 min. The kinetics of inactivation were examined under conditions where the Schiff base was reduced before assay. Inactivation was apparently first-order. The pseudo-first-order rate constant, kapp., showed a hyperbolic dependence upon the concentration of pyridoxal 5'-phosphate, suggesting that the enzyme first formed a non-covalent complex with the reagent, modification of a lysine then proceeding within this complex. Inactivation of the enzyme by pyridoxal was 20 times slower than that by pyridoxal 5'-phosphate, indicating that the phosphate group was important in forming the initial complex. Partial protection against pyridoxal phosphate was provided by the leading substrate, carbamoyl phosphate, and nearly complete protection was provided by the bisubstrate analogue, N-phosphonoacetyl-L-aspartate, and the ligand-pair carbamoyl phosphate plus succinate. Steady-state kinetic studies, under conditions that minimized inactivation, showed that pyridoxal 5'-phosphate was also a competitive inhibitor with respect to the leading substrate, carbamoyl phosphate. Pyridoxal 5'-phosphate therefore appears to be an active-site-directed reagent. A sample of the enzyme containing one reduced pyridoxyl group per chain was digested with trypsin, and the labelled peptide was isolated and shown to contain a single pyridoxyl-lysine residue. Partial sequencing around the labelled lysine showed little homology with the sequence surrounding lysine-84, an active-centre residue of the catalytic subunit of aspartate transcarbamoylase from Escherichia coli, whose reaction with pyridoxal 5'-phosphate shows many similarities to the results described in the present paper. Arguably the reactive lysine is conserved between the two enzymes whereas the residues immediately surrounding the lysine are not. The same conclusion has been drawn in a comparison of reactive histidine residues in the two enzymes [Cole & Yon (1986) Biochemistry 25, 7168-7174].     

Role of arginine residues in the active site of the membrane-bound lytic transglycosylase B from Pseudomonas aeruginosa

Lytic transglycosylases cleave the beta-(1-->4)-glycosidic bond in the bacterial cell wall heteropolymer peptidoglycan between the N-acetylmuramic acid (MurNAc) and N-acetylglucosamine (GlcNAc) residues with the concomitant formation of a 1,6-anhydromuramoyl residue. On the basis of both sequence alignments with and structural considerations of soluble lytic transglycosylase Slt35 from Escherichia coli, four residues were predicted to be involved in substrate binding at the -1 subsite in the soluble derivative of Pseudomonas aeruginosa membrane-bound lytic transglycosylase MltB. These residues were targeted for site-specific replacement, and the effect on substrate binding and catalysis was determined. The residues Arg187 and Arg188, believed to be involved in binding the stem peptide on MurNAc, were shown to play an important role in substrate binding, as evidenced by peptidoglycan affinity assays and SUPREX analysis using MurNAc-dipeptide as ligand. The Michaelis-Menten parameters were determined for the respective mutants using insoluble peptidoglycan as substrate. In addition to affecting the steady-state binding of ligand to enzyme, as indicated by increases in K(M) values, significant decreases in k(cat) values suggested that replacement of either Arg187 and Arg188 with alanine perturbed the stabilization of both the transition state(s) and reaction intermediate. Thus, it appears that Arg187 and Arg188 are vital for proper orientation of the substrate in the active site, and furthermore this supports the proposed role of the stem peptide at binding subsite -2 in catalysis. Replacement of Gln100, a residue that would appear to interact with the N-acetyl group on MurNAc, did not show any changes in substrate affinity or activity.     

The substrate activation process in the catalytic reaction of Escherichia coli aromatic amino acid aminotransferase

Aromatic amino acid aminotransferase is active toward both aromatic and dicarboxylic amino acids, and the mechanism for this dual substrate recognition has been an issue in the enzymology of this enzyme. Here we show that, in the reactions with aromatic and dicarboxylic ligands, the pK(a) of the Schiff base formed between the coenzyme pyridoxal 5'-phosphate and Lys258 or the substrate increases successively from 6.6 in the unliganded enzyme to approximately 8.8 in the Michaelis complex and to >10.5 in the external Schiff base complex. Mutations of Arg292 and Arg386 to Leu, which mimic neutralization of the positive charges of the two arginine residues by the ligand carboxylate groups, increased the Schiff base pK(a) by 0.1 and 0.7 unit, respectively. In contrast to these moderate effects of the Arg mutations, the cleavage of the Lys258 side chain of the Schiff base, which was brought about by preparing a mutant enzyme in which Lys258 was changed to Ala and the Schiff base was reconstituted with methylamine, produced the Schiff base pK(a) value of 10.2, that being 3.6 units higher than that of the wild-type enzyme. The observation indicates that the Schiff base pK(a) in the enzyme is lowered by the torsion around the C4-C4' axis of the Schiff base and suggests that the pK(a) is mainly controlled by changing the torsion angle during the course of catalysis. This mechanism, first observed for the reaction of aspartate aminotransferase with aspartate [Hayashi, H., Mizuguchi, H., and Kagamiyama, H. (1998) Biochemistry 37, 15076-15085], does not require the electrostatic contribution from the omega-carboxylate group of the substrate, and can explain why in aromatic amino acid aminotransferase the aromatic substrates can increase the Schiff base pK(a) during catalysis to the same extent as the dicarboxylic substrates. This is the first example in which the torsion pK(a) coupling of the pyridoxal 5'-phosphate Schiff base has been demonstrated in pyridoxal enzymes other than aspartate aminotransferase, and suggests the generality of the mechanism in the catalysis of aminotransferases related to aspartate aminotransferase.     

Mechanism of the Class I KDPG aldolase

In vivo, 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase catalyzes the reversible, stereospecific retro-aldol cleavage of KDPG to pyruvate and D-glyceraldehyde-3-phosphate. The enzyme is a lysine-dependent (Class I) aldolase that functions through the intermediacy of a Schiff base. Here, we propose a mechanism for this enzyme based on crystallographic studies of wild-type and mutant aldolases. The three dimensional structure of KDPG aldolase from the thermophile Thermotoga maritima was determined to 1.9A. The structure is the standard alpha/beta barrel observed for all Class I aldolases. At the active site Lys we observe clear density for a pyruvate Schiff base. Density for a sulfate ion bound in a conserved cluster of residues close to the Schiff base is also observed. We have also determined the structure of a mutant of Escherichia coli KDPG aldolase in which the proposed general acid/base catalyst has been removed (E45N). One subunit of the trimer contains density suggesting a trapped pyruvate carbinolamine intermediate. All three subunits contain a phosphate ion bound in a location effectively identical to that of the sulfate ion bound in the T. maritima enzyme. The sulfate and phosphate ions experimentally locate the putative phosphate binding site of the aldolase and, together with the position of the bound pyruvate, facilitate construction of a model for the full-length KDPG substrate complex. The model requires only minimal positional adjustments of the experimentally determined covalent intermediate and bound anion to accommodate full-length substrate. The model identifies the key catalytic residues of the protein and suggests important roles for two observable water molecules. The first water molecule remains bound to the enzyme during the entire catalytic cycle, shuttling protons between the catalytic glutamate and the substrate. The second water molecule arises from dehydration of the carbinolamine and serves as the nucleophilic water during hydrolysis of the enzyme-product Schiff base. The second water molecule may also mediate the base-catalyzed enolization required to form the carbon nucleophile, again bridging to the catalytic glutamate. Many aspects of this mechanism are observed in other Class I aldolases and suggest a mechanistically and, perhaps, evolutionarily related family of aldolases distinct from the N-acetylneuraminate lyase (NAL) family.     

Strain is more important than electrostatic interaction in controlling the pKa of the catalytic group in aspartate aminotransferase

Systematic single and multiple replacement studies have been applied to Escherichia coli aspartate aminotransferase to probe the electrostatic effect of the two substrate-binding arginine residues, Arg292 and Arg386, and the structural effect of the pyridoxal 5'-phosphate-Asn194-Arg386 hydrogen-bond linkage system (PLP-N-R) on the pK(a) value of the Schiff base formed between pyridoxal 5'-phosphate (PLP) and Lys258. The electrostatic effects of the two arginine residues cannot be assessed by simple mutational studies of the residues. PLP-N-R lowers the pK(a) value of the PLP-Lys258 Schiff base by keeping it in the distorted conformation, which is unfavorable for protonation. Mutation of Arg386 eliminates its hydrogen bond with Asn194 and partially disrupts PLP-N-R, thereby relaxing the strain of the Schiff base. On the other hand, mutation of Arg292, the large domain residue that interacts with the small domain residue Asp15, makes the domain opening easier. Because PLP-N-R lies between the two domains, the domain opening increases the strain of the Schiff base. Therefore, the true electrostatic effects of Arg292 and Arg386 could be derived from mutational analysis of the enzyme in which PLP-N-R had been completely disrupted by the Asn194Ala mutation. Through the analyses, we could dissect the electrostatic and structural effects of the arginine mutations on the Schiff base pK(a). The positive charges of the two arginine residues and the PLP-N-R-mediated strain of the Schiff base lower the Schiff base pK(a) by 0.7 and 1.7, respectively. Thus, the electrostatic effect of the arginine residues is not as strong as has historically been thought, and this finding substantiates our recent finding that the imine-pyridine torsion of the Schiff base is the primary determinant (2.8 unit decrease) of the extremely low pK(a) value of the Schiff base [Hayashi, H., Mizuguchi, H., and Kagamiyama, H. (1998) Biochemistry 37, 15076-15085].     

Snapshots of catalysis: the structure of fructose-1,6-(bis)phosphate aldolase covalently bound to the substrate dihydroxyacetone phosphate.

Fructose-1,6-bis(phosphate) aldolase is an essential glycolytic enzyme found in all vertebrates and higher plants that catalyzes the cleavage of fructose 1,6-bis(phosphate) (Fru-1,6-P(2)) to glyceraldehyde 3-phosphate and dihydroxyacetone phosphate (DHAP). Mutations in the aldolase genes in humans cause hemolytic anemia and hereditary fructose intolerance. The structure of the aldolase-DHAP Schiff base has been determined by X-ray crystallography to 2.6 A resolution (R(cryst) = 0.213, R(free) = 0.249) by trapping the catalytic intermediate with NaBH(4) in the presence of Fru-1,6-P(2). This is the first structure of a trapped covalent intermediate for this essential glycolytic enzyme. The structure allows the elucidation of a comprehensive catalytic mechanism and identification of a conserved chemical motif in Schiff-base aldolases. The position of the bound DHAP relative to Asp33 is consistent with a role for Asp33 in deprotonation of the C4-hydroxyl leading to C-C bond cleavage. The methyl side chain of Ala31 is positioned directly opposite the C3-hydroxyl, sterically favoring the S-configuration of the substrate at this carbon. The "trigger" residue Arg303, which binds the substrate C6-phosphate group, is a ligand to the phosphate group of DHAP. The observed movement of the ligand between substrate and product phosphates may provide a structural link between the substrate cleavage and the conformational change in the C-terminus associated with product release. The position of Glu187 in relation to the DHAP Schiff base is consistent with a role for the residue in protonation of the hydroxyl group of the carbinolamine in the dehydration step, catalyzing Schiff-base formation. The overlay of the aldolase-DHAP structure with that of the covalent enzyme-dihydroxyacetone structure of the mechanistically similar transaldolase and KDPG aldolase allows the identification of a conserved Lys-Glu dyad involved in Schiff-base formation and breakdown. The overlay highlights the fact that Lys146 in aldolase is replaced in transaldolase with Asn35. The substitution in transaldolase stabilizes the enamine intermediate required for the attack of the second aldose substrate, changing the chemistry from aldolase to transaldolase.     

Repair activities of 8-oxoguanine DNA glycosylase from Archaeoglobus fulgidus, a hyperthermophilic archaeon.

Oxidative DNA damage is caused by reactive oxygen species formed in cells as by products of aerobic metabolism or of oxidative stress. The 8-oxoguanine (8-oxoG) DNA glycosylase from Archaeoglobus fulgidus (Afogg), which excises an oxidatively-damaged form of guanine, was overproduced in Escherichia coli, purified and characterized. A. fulgidus is a sulfate-reducing archaeon, which grows at between 60 and 95 degrees C, with an optimum growth at 83 degrees C. The Afogg enzyme has both DNA glycosylase and apurinic/apyrimidinic (AP) lyase activities, with the latter proceeding through a Schiff base intermediate. As expected for a protein from a hyperthermophilic organism, the enzyme activity is optimal near pH 8.5 and 60 degrees C, denaturing at 80 degrees C, and is thermally stable at high levels of salt (500mM). The Afogg protein efficiently cleaves oligomers containing 8-oxoG:C and 8-oxoG:G base pairs, and is less effective on oligomers containing 8-oxoG:T and 8-oxoG:A mispairs. While the catalytic action mechanism of Afogg protein is likely similar to the human Ogg1 (hOgg1), the DNA recognition mechanism and the basis for 8-oxoG substrate specificity of Afogg differ from that of hOgg.     

Biochemical characterization of a thermostable cysteine synthase from Geobacillus stearothermophilus V

The cysK gene encoding a cysteine synthase of Geobacillus stearothermophilus V was overexpressed in E. coli and the recombinant protein was purified and characterized. The enzyme is a thermostable homodimer (32 kDa/monomer) belonging to the beta family of pyridoxal phosphate (PLP)-dependent enzymes. UV-visible spectra showed absorption bands at 279 and 410 nm. The band at 279 nm is due to tyrosine residues as the enzyme lacks tryptophan. The 410 nm band represents absorption of the coenzyme bound as a Schiff base to a lysine residue of the protein. Fluorescence characteristics of CysK's Schiff base were influenced by temperature changes suggesting different local structures at the cofactor binding site. The emission of the Schiff base allowed the determination of binding constants for products at both 20 degrees C and 50 degrees C. At 50 degrees C and in the absence of sulphide the enzyme catalyzes the decomposition of O-acetyl-l-serine to pyruvate and ammonia. At 20 degrees C, however, a stable alpha-aminoacrylate intermediate is formed.     

Crystal structure of phenylalanine ammonia lyase: multiple helix dipoles implicated in catalysis

The first three-dimensional structure of phenylalanine ammonia lyase (PAL) has been determined at 2.1 A resolution for PAL from Rhodosporidium toruloides. The enzyme is structurally similar to the mechanistically related histidine ammonia lyase (HAL), with PAL having an additional approximately 160 residues extending from the common fold. We propose that catalysis (including lowering the pK(a) of nonacidic C3 of l-phenylalanine for an E1cb mechanism) is potentially governed by dipole moments of seven alpha helices associated with the PAL active site (six positive poles and one negative pole). Cofactor 3,5-dihydro-5-methylidene-4H-imidazol-4-one (MIO) resides atop the positive poles of three helices, for increasing its electrophilicity. The helix dipoles appear fully compatible with a model of phenylalanine docked in the active site of PAL having the first covalent bond formed between the amino group of substrate and the methylidene group of MIO: 12 highly conserved residues (near the N termini of helices for enhancing function) are poised to serve roles in substrate recognition, MIO activation, product separation, proton donation, or polarizing electrons from the phenyl ring of substrate for activation of C3; and a highly conserved His residue (near the C terminus of the one helix that directs its negative pole toward the active site to increase the residue's basicity) is positioned to act as a general base, abstracting the pro-S hydrogen from C3 of substrate. A similar mechanism is proposed for HAL, which has a similar disposition of seven alpha helices and similar active-site residues. The helix dipoles appear incompatible with a proposed mechanism that invokes a carbocation intermediate.     

The active site of the Escherichia coli MutY DNA adenine glycosylase.

Escherichia coli MutY is an adenine DNA glycosylase active on DNA substrates containing A/G, A/C, or A/8-oxoG mismatches. Although MutY can form a covalent intermediate with its DNA substrates, its possession of 3' apurinic lyase activity is controversial. To study the reaction mechanism of MutY, the conserved Asp-138 was mutated to Asn and the reactivity of this mutant MutY protein determined. The glycosylase activity was completely abolished in the D138N MutY mutant. The D138N mutant and wild-type MutY protein also possessed different DNA binding activities with various mismatches. Several lysine residues were identified in the proximity of the active site by analyzing the imino-covalent MutY-DNA intermediate. Mutation of Lys-157 and Lys-158 both individually and combined, had no effect on MutY activities but the K142A mutant protein was unable to form Schiff base intermediates with DNA substrates. However, the MutY K142A mutant could still bind DNA substrates and had adenine glycosylase activity. Surprisingly, the K142A mutant MutY, but not the wild-type enzyme, could promote a beta/delta-elimination on apurinic DNA. Our results suggest that Asp-138 acts as a general base to deprotonate either the epsilon-amine group of Lys-142 or to activate a water molecule and the resulting apurinic DNA then reacts with Lys-142 to form the Schiff base intermediate with DNA. With the K142A mutant, Asp-138 activates a water molecule to attack the C1' of the adenosine; the resulting apurinic DNA is cleaved through beta/delta-elimination without Schiff base formation.     

A structural and mechanistic comparison of pyridoxal 5'-phosphate dependent decarboxylase and transaminase enzymes.

Stereochemical studies of three pyridoxal phosphate dependent decarboxylases and serine hydroxymethyltransferase have allowed the dispositions of conjugate acids that operate at the C alpha and C-4' positions of intermediate quinoids to be determined. Kinetic work with the decarboxylase group has determined that two different acids are involved, a monoprotic acid and a polyprotic acid. The use of solvent kinetic isotope effects allowed the resolution of chemical steps in the reaction coordinate profile for decarboxylation and abortive transamination and pH-sensitivities gave the molecular pKa of the monoprotic base. Thus the epsilon-ammonium group of the internal aldimine-forming lysine residue operates at C-4'-si-face of the coenzyme and the imidazolium side chain of an active site histidine residue protonates at C alpha from the 4'-si-face. Histidine serves two other functions, as a base in generating nitrogen nucleophiles during both transaldimination processes and as a binding group for the alpha-carboxyl group of substrates. The latter role for histidine was determined by comparison of the sequences for decarboxylase active site tetrapeptides (e.g. -S-X-H-K-) with that for aspartate aminotransferase (e.g. -S-X-A-K-) where it was known, from X-ray studies, that the serine and lysine residues interact with the coenzyme. By using the Dunathan Postulate, the conformation of the external aldimine was modified, and without changing the tetrapeptide conformation, the alanine residue was altered to a histidine. This model for the active site of a pyridoxal dependent decarboxylase was consistent with all available stereochemical and mechanistic data.(ABSTRACT TRUNCATED AT 250 WORDS)     

Decarboxylases involved in polyamine biosynthesis and their inactivation by nitric oxide

Polyamines are ubiquitous cellular components that are involved in normal and neoplastic growth. Polyamine biosynthesis is very highly regulated in mammalian cells by the activities of two key decarboxylases acting on ornithine and S-adenosylmethionine. Recent studies, which include crystallographic analysis of the recombinant human proteins, have provided a detailed knowledge of their structure and function. Ornithine decarboxylase is a PLP-requiring decarboxylase, whereas S-adenosylmethionine decarboxylase (AdoMetDC) contains a covalently bound pyruvate prosthetic group. Both enzymes have a key cysteine residue, which is involved in protonation of the Schiff base intermediate C(alpha) to form the product. These residues, Cys360 in ornithine decarboxylase (ODC) and Cys82 in AdoMetDC, react readily with nitric oxide (NO), which is therefore a potent inactivator of polyamine synthesis. The inactivation of these enzymes may mediate some of the antiproliferative actions of NO.     

Mutation at active site lysine 212 to arginine uncouples the glycosylase activity from the lyase activity of human endonuclease III.

The human endonuclease III (hNTH1) is an important DNA glycosylase with associated abasic lyase activity. We previously demonstrated that the K212Q mutant was totally inactive, while the K212R mutant had reduced DNA glycosylase/lyase activity and could form a covalent complex with the substrate DNA upon reduction. We further characterized the biochemical properties of this K212R mutant protein. NH2- (N-) terminal sequencing in combination with mass spectrometry of the peptide-DNA adduct suggested that "opportunistic" lysine(s) in the lysine-rich N-terminal tail formed a Schiff base which might result in beta-elimination. However, simultaneous substitution of Lys-75 with Gln and deletion of first 72 residues in the N-terminal tail could not cause further alteration in the glycosylase reaction or beta-elimination event. Nonetheless, the time kinetics of K212R and its subsequent mutants showed glycosylase activity without any detectable AP-lyase activity during the first 10 min of the reaction. These results suggest that a single point mutation at the active site (K212R) uncoupled the glycosylase activity from the lyase activity. We propose that the uncoupled reaction carried out by K212R is a result of direct attack either by the nonionized form of the guanidino group of arginine which forms an unstable Schiff base that hydrolyzes prior to the beta-elimination event or by hydroxide ion to cleave the glycosylic bond. In either case this reaction is followed by a secondary beta-elimination event performed by random lysine residues primarily from the N-terminal tail region.     

New superfamily members identified for Schiff-base enzymes based on verification of catalytically essential residues

Enzymes that utilize a Schiff-base intermediate formed with their substrates and that share the same alpha/beta barrel fold comprise a mechanistically diverse superfamily defined in the SCOPS database as the class I aldolase family. The family includes the "classical" aldolases fructose-1,6-(bis)phosphate (FBP) aldolase, transaldolase, and 2-keto-3-deoxy-6-phosphogluconate aldolase. Moreover, the N-acetylneuraminate lyase family has been included in the class I aldolase family on the basis of similar Schiff-base chemistry and fold. Herein, we generate primary sequence identities based on structural alignment that support the homology and reveal additional mechanistic similarities beyond the common use of a lysine for Schiff-base formation. The structural and mechanistic correspondence comprises the use of a catalytic dyad, wherein a general acid/base residue (Glu, Tyr, or His) involved in Schiff-base chemistry is stationed on beta-strand 5 of the alpha/beta barrel. The role of the acid/base residue was probed by site-directed mutagenesis and steady-state and pre-steady-state kinetics on a representative member of this family, FBP aldolase. The kinetic results are consistent with the participation of this conserved residue or position in the protonation of the carbinolamine intermediate and dehydration of the Schiff base in FBP aldolase and, by analogy, the class I aldolase family.     

15N and 13C NMR studies of ligands bound to the 280,000-dalton protein porphobilinogen synthase elucidate the structures of enzyme-bound product and a Schiff base intermediate

Porphobilinogen synthase (PBGS) catalyzes the asymmetric condensation of two molecules of 5-aminolevulinic acid (ALA). Despite the 280,000-dalton size of PBGS, much can be learned about the reaction mechanism through 13C and 15N NMR. To our knowledge, these studies represent the largest protein complex for which individual nuclei have been characterized by 13C or 15N NMR. Here we extend our 13C NMR studies to PBGS complexes with [3,3-2H2,3-13C]ALA and report 15N NMR studies of [15N]ALA bound to PBGS. As in our previous 13C NMR studies, observation of enzyme-bound 15N-labeled species was facilitated by deuteration at nitrogens that are attached to slowly exchanging hydrogens. For holo-PBGS at neutral pH, the NMR spectra reflect the structure of the enzyme-bound product porphobilinogen (PBG), whose chemical shifts are uniformly consistent with deprotonation of the amino group whose solution pKa is 11. Despite this local environment, the protons of the amino group are in rapid exchange with solvent (kexchange greater than 10(2) s-1). For methyl methanethiosulfonate (MMTS) modified PBGS, the NMR spectra reflect the chemistry of an enzyme-bound Schiff base intermediate that is formed between C4 of ALA and an active-site lysine. The 13C chemical shift of [3,3-2H2,3-13C]ALA confirms that the Schiff base is an imine of E stereochemistry. By comparison to model imines formed between [15N]ALA and hydrazine or hydroxylamine, the 15N chemical shift of the enzyme-bound Schiff base suggests that the free amino group is an environment resembling partial deprotonation; again the protons are in rapid exchange with solvent. Deprotonation of the amino group would facilitate formation of a Schiff base between the amino group of the enzyme-bound Schiff base and C4 of the second ALA substrate. This is the first evidence supporting carbon-nitrogen bond formation as the initial site of interaction between the two substrate molecules.     

A conserved glutamate residue exhibits multifunctional catalytic roles in D-fructose-1,6-bisphosphate aldolases.

The aldolase catalytic cycle consists of a number of proton transfers that interconvert covalent enzyme intermediates. Glu-187 is a conserved amino acid that is located in the mammalian fructose-1,6-bisphosphate aldolase active site. Its central location, within hydrogen bonding distance of three other conserved active site residues: Lys-146, Glu-189, and Schiff base-forming Lys-229, makes it an ideal candidate for mediating proton transfers. Point mutations, Glu-187--> Gln, Ala, which would inhibit proton transfers significantly, compromise activity. Trapping of enzymatic intermediates in Glu-187 mutants defines a proton transfer role for Glu-187 in substrate cleavage and Schiff base formation. Structural data show that loss of Glu-187 negative charge results in hydrogen bond formation between Lys-146 and Lys-229 consistent with a basic pK(a) for Lys-229 in native enzyme and supporting nucleophilic activation of Lys-229 by Glu-187 during Schiff base formation. The crystal structures also substantiate Glu-187 and Glu-189 as present in ionized form in native enzyme, compatible with their role of catalyzing proton exchange with solvent as indicated from solvent isotope effects. The proton exchange mechanism ensures Glu-187 basicity throughout the catalytic cycle requisite for mediating proton transfer and electrostatic stabilization of ketamine intermediates. Glutamate general base catalysis is a recurrent evolutionary feature of Schiff base0forming aldolases.     

Survey of catalytic residues and essential roles of glutamate-alpha170 and aspartate-alpha335 in coenzyme B12-dependent diol dehydratase

The importance of each active-site residue in adenosylcobalamin-dependent diol dehydratase of Klebsiella oxytoca was estimated using mutant enzymes in which one of the residues interacting with substrate and/or K(+) was mutated to Ala or another amino acid residue. The Ealpha170A and Dalpha335A mutants were totally inactive, and the Halpha143A mutant showed only a trace of activity, indicating that Glu-alpha170, Asp-alpha335, and His-alpha143 are catalytic residues. The Qalpha141A, Qalpha296A, and Salpha362A mutants showed partial activity. It was suggested from kinetic parameters that Gln-alpha296 is important for substrate binding and Gln-alpha296 and Gln-alpha141 for preventing the enzyme from mechanism-based inactivation. The Ealpha221A, Ealpha170H, and Dalpha335A did not form the (alphabetagamma)(2) complex, suggesting that these mutations indirectly disrupt subunit contacts. Among other Glu-alpha170 and Asp-alpha335 mutants, Ealpha170D and Ealpha170Q were 2.2 +/- 0.3% and 0.02% as active as the wild-type enzyme, respectively, whereas Dalpha335N was totally inactive. Kinetic analysis indicated that the presence and the position of a carboxyl group in the residue alpha170 are essential for catalysis as well as for the continuous progress of catalytic cycles. It was suggested that the roles of Glu-alpha170 and Asp-alpha335 are to participate in the binding of substrate and intermediates and keep them appropriately oriented and to function as a base in the dehydration of the 1,1-diol intermediate. In addition, Glu-alpha170 seems to stabilize the transition state for the hydroxyl group migration from C2 to C1 by accepting the proton of the spectator hydroxyl group on C1.