A Historical Perspective for the Catalytic Reaction Mechanism of Glycosidase; So As to Bring about Breakthrough in Confusing Situation

Various catalytic reaction models have been proposed as the reaction mechanisms of glycosidases, but a reasonable and unitary model capable of interpreting both “inverting” and “retaining” glycosidase reactions remains to be established. As for the models proposed to date, the nucleophilic displacement mechanism and the oxocarbenium ion intermediate mechanism are widely known, but recently the former is widely accepted, and so the general tendency of world opinion appears to favor it. This reaction model, however, is considered to comprise some inconsistencies that cannot be neglected from the viewpoint of reactivity in organic chemistry. While the nucleophilic displacement mechanism is often applied to reactions of glycosidases, it appears unlikely that such reactions actually occur. This review argues that the oxocarbenium ion intermediate reaction mechanism is more rational than the nucleophilic displacement reaction mechanism, as the action mode of glycosidases and related enzymes.     

LacZ β‐galactosidase: Structure and function of an enzyme of historical and molecular biological importance

This review provides an overview of the structure, function, and catalytic mechanism of lacZ β‐galactosidase. The protein played a central role in Jacob and Monod's development of the operon model for the regulation of gene expression. Determination of the crystal structure made it possible to understand why deletion of certain residues toward the amino‐terminus not only caused the full enzyme tetramer to dissociate into dimers but also abolished activity. It was also possible to rationalize α‐complementation, in which addition to the inactive dimers of peptides containing the “missing” N‐terminal residues restored catalytic activity. The enzyme is well known to signal its presence by hydrolyzing X‐gal to produce a blue product. That this reaction takes place in crystals of the protein confirms that the X‐ray structure represents an active conformation. Individual tetramers of β‐galactosidase have been measured to catalyze 38,500 ± 900 reactions per minute. Extensive kinetic, biochemical, mutagenic, and crystallographic analyses have made it possible to develop a presumed mechanism of action. Substrate initially binds near the top of the active site but then moves deeper for reaction. The first catalytic step (called galactosylation) is a nucleophilic displacement by Glu537 to form a covalent bond with galactose. This is initiated by proton donation by Glu461. The second displacement (degalactosylation) by water or an acceptor is initiated by proton abstraction by Glu461. Both of these displacements occur via planar oxocarbenium ion‐like transition states. The acceptor reaction with glucose is important for the formation of allolactose, the natural inducer of the lac operon.     

Crystal structures of Geobacillus stearothermophilus alpha-glucuronidase complexed with its substrate and products: mechanistic implications

Alpha-glucuronidases cleave the alpha-1,2-glycosidic bond between 4-O-methyl-d-glucuronic acid and short xylooligomers as part of the hemicellulose degradation system. To date, all of the alpha-glucuronidases are classified as family 67 glycosidases, which catalyze the hydrolysis via the investing mechanism. Here we describe several high resolution crystal structures of the alpha-glucuronidase (AguA) from Geobacillus stearothermophilus, in complex with its substrate and products. In the complex of AguA with the intact substrate, the 4-O-methyl-d-glucuronic acid sugar ring is distorted into a half-chair conformation, which is closer to the planar conformation required for the oxocarbenium ion-like transition state structure. In the active site, a water molecule is coordinated between two carboxylic acids, in an appropriate position to act as a nucleophile. From the structural data it is likely that two carboxylic acids, Asp(364) and Glu(392), activate together the nucleophilic water molecule. The loop carrying the catalytic general acid Glu(285) cannot be resolved in some of the structures but could be visualized in its "open" and "closed" (catalytic) conformations in other structures. The protonated state of Glu(285) is presumably stabilized by its proximity to the negative charge of the substrate, representing a new variation of substrate-assisted catalysis mechanism.     

Serine hydroxymethyltransferase: role of glu75 and evidence that serine is cleaved by a retroaldol mechanism

Serine hydroxymethyltransferase (SHMT) catalyzes the reversible interconversion of serine and glycine with tetrahydrofolate serving as the one-carbon carrier. SHMT also catalyzes the folate-independent retroaldol cleavage of allothreonine and 3-phenylserine and the irreversible conversion of 5,10-methenyltetrahydrofolate to 5-formyltetrahydrofolate. Studies of wild-type and site mutants of SHMT have failed to clearly establish the mechanism of this enzyme. The cleavage of 3-hydroxy amino acids to glycine and an aldehyde occurs by a retroaldol mechanism. However, the folate-dependent cleavage of serine can be described by either the same retroaldol mechanism with formaldehyde as an enzyme-bound intermediate or by a nucleophilic displacement mechanism in which N5 of tetrahydrofolate displaces the C3 hydroxyl of serine, forming a covalent intermediate. Glu75 of SHMT is clearly involved in the reaction mechanism; it is within hydrogen bonding distance of the hydroxyl group of serine and the formyl group of 5-formyltetrahydrofolate in complexes of these species with SHMT. This residue was changed to Leu and Gln, and the structures, kinetics, and spectral properties of the site mutants were determined. Neither mutation significantly changed the structure of SHMT, the spectral properties of its complexes, or the kinetics of the retroaldol cleavage of allothreonine and 3-phenylserine. However, both mutations blocked the folate-dependent serine-to-glycine reaction and the conversion of methenyltetrahydrofolate to 5-formyltetrahydrofolate. These results clearly indicate that interaction of Glu75 with folate is required for folate-dependent reactions catalyzed by SHMT. Moreover, we can now propose a promising modification to the retroaldol mechanism for serine cleavage. As the first step, N5 of tetrahydrofolate makes a nucleophilic attack on C3 of serine, breaking the C2-C3 bond to form N5-hydroxymethylenetetrahydrofolate and an enzyme-bound glycine anion. The transient formation of formaldehyde as an intermediate is possible, but not required. This mechanism explains the greatly enhanced rate of serine cleavage in the presence of folate, and avoids some serious difficulties presented by the nucleophilic displacement mechanism involving breakage of the C3-OH bond.     

Characterization of a novel thiosulfate-forming enzyme isolated from Desulfovibrio vulgaris.

An enzyme that formed thiosulfate from bisulfite and trithionate was purified from extracts of Desulfovibrio vulgaris. This enzyme, designated as "thiosulfate-forming" enzyme, required the presence of both bisulfite and trithionate. Various 35S-labeling studies showed that thiosulfate was formed from bisulfite and the inner sulfur atom of trithionate. This involved a nucleophilic attack by the bisulfite ion, resulting in the displacement of the two outer sulfonate groups of trithionate that recycled to participate as free bisulfite in subsequent reactions. This reaction required a reduction, presumably by a concerted mechanism with thiosulfate formation. The natural electron carrier cytochrome c3 participated in this reductive formation of thiosulfate. This reaction was coupled to the bisulfite reductase-catalyzed reaction, which resulted in the reconstruction of a thiosulfate-forming pathway from bisulfite.     

Detailed kinetic analysis of a family 52 glycoside hydrolase: a beta-xylosidase from Geobacillus stearothermophilus

Geobacillus stearothermophilus T-6 encodes for a beta-xylosidase (XynB2) from family 52 of glycoside hydrolases that was previously shown to hydrolyze its substrate with net retention of the anomeric configuration. XynB2 significantly prefers substrates with xylose as the glycone moiety and exhibits a typical bell-shaped pH dependence curve. Binding properties of xylobiose and xylotriose to the active site were measured using isothermal titration calorimetry (ITC). Binding reactions were enthalpy driven with xylobiose binding more tightly than xylotriose to the active site. The kinetic constants of XynB2 were measured for the hydrolysis of a variety of aryl beta-D-xylopyranoside substrates bearing different leaving groups. The Br?nsted plot of log k(cat) versus the pK(a) value of the aglycon leaving group reveals a biphasic relationship, consistent with a double-displacement mechanism as expected for retaining glycoside hydrolases. Hydrolysis rates for substrates with poor leaving groups (pK(a) > 8) vary widely with the aglycon reactivity, indicating that, for these substrates, the bond cleavage is rate limiting. However, no such dependence is observed for more reactive substrates (pK(a) < 8), indicating that in this case hydrolysis of the xylosyl-enzyme intermediate is rate limiting. Secondary kinetic isotope effects suggest that the intermediate breakdown proceeds with modest oxocarbenium ion character at the transition state, and bond cleavage proceeds with even lower oxocarbenium ion character. Inhibition studies with several gluco analogue inhibitors could be measured since XynB2 has low, yet sufficient, activity toward 4-nitrophenyl beta-D-glucopyranose. As expected, inhibitors mimicking the proposed transition state structure, such as 1-deoxynojirimycin, bind with much higher affinity to XynB2 than ground state inhibitors.     

Analysis of glycosidase-catalyzed transglycosylation reaction using probabilistic model

General mechanism of transglycosylation reaction by glycosidases contains branched paths to form and destroy the glycosylated intermediate. The probabilistic model was applied for the simulation and analysis of the transglycosylation mechanism. The model is composed of a single enzyme molecule and finite amounts of substrates and water molecules mimicking the possible smallest enzyme-catalyzed reaction system in a microcompartment. Using random numbers and probabilities, progress of distribution of reactants and products can be simulated and predicted with minimum adjustable parameters. Experimental data of beta-xylosidase and beta-glucosidase reactions were quantitatively analyzed with the simple scheme. Since the algorithm and simulation procedures are simple, the model is applicable to related complicated enzyme mechanisms containing many branched reaction paths.     

Production of 1,5-anhydro-d-fructose by an α-glucosidase belonging to glycoside hydrolase family 31

α-1,4-Glucan lyases [glycoside hydrolase family (GH) 31] catalyze an elimination reaction to form 1,5-anhydro-d-fructose (AF), while GH31 α-glucosidases normally catalyze a hydrolytic reaction. We determined that a small amount of AF was produced by GH31 Aspergillus niger α-glucosidase from maltooligosaccharides by elimination reaction, likely via an oxocarbenium ion intermediate.     

Analysis of solvent nucleophile isotope effects: evidence for concerted mechanisms and nucleophilic activation by metal coordination in nonenzymatic and ribozyme-catalyzed phosphodiester hydrolysis

Heavy atom isotope effects are a valuable tool for probing chemical and enzymatic reaction mechanisms; yet, they are not widely applied to examine mechanisms of nucleophilic activation. We developed approaches for analyzing solvent (18)O nucleophile isotope effects ((18)k(nuc)) that allow, for the first time, their application to hydrolysis reactions of nucleotides and nucleic acids. Here, we report (18)k(nuc) for phosphodiester hydrolysis catalyzed by Mg(2+) and by the Mg(2+)-dependent RNase P ribozyme and deamination by the Zn(2+)-dependent protein enzyme adenosine deaminase (ADA). Because ADA incorporates a single solvent molecule into the product inosine, this reaction can be used to monitor solvent (18)O/(16)O ratios in complex reaction mixtures. This approach, combined with new methods for analysis of isotope ratios of nucleotide phosphates by whole molecule mass spectrometry, permitted determination of (18)k(nuc) for hydrolysis of thymidine 5'-p-nitrophenyl phosphate and RNA cleavage by the RNase P ribozyme. For ADA, an inverse (18)k(nuc) of 0.986 +/- 0.001 is observed, reflecting coordination of the nucleophile by an active site Zn(2+) ion and a stepwise mechanism. In contrast, the observed (18)k(nuc) for phosphodiester reactions were normal: 1.027 +/- 0.013 and 1.030 +/- 0.012 for the Mg(2+)- and ribozyme-catalyzed reactions, respectively. Such normal effects indicate that nucleophilic attack occurs in the rate-limiting step for these reactions, consistent with concerted mechanisms. However, these magnitudes are significantly less than the (18)k(nuc) observed for nucleophilic attack by hydroxide (1.068 +/- 0.007), indicating a "stiffer" bonding environment for the nucleophile in the transition state. Kinetic analysis of the Mg(2+)-catalyzed reaction indicates that a Mg(2+)-hydroxide complex is the catalytic species; thus, the lower (18)k(nuc), in large part, reflects direct metal ion coordination of the nucleophilic oxygen. A similar value for the RNase P ribozyme catalyzed reaction provides support for nucleophilic activation by metal ion catalysis.     

Computational modeling of the rate limiting step in low molecular weight protein tyrosine phosphatase.

Hydrolysis of the phosphoenzyme intermediate is the second and rate limiting step of the reaction catalyzed by the protein tyrosine phosphatases (PTPs). The cysteinyl phosphate thioester bond is cleaved by nucleophilic displacement where an active site water molecule attacks the phosphorus atom. Starting from the crystal structure of the low molecular weight PTP, we study the energetics of this reaction utilizing the empirical valence bond method in combination with molecular dynamics and free energy perturbation simulations. The reactions of the wild-type as well as the D129A and C17S mutants are modeled. For the D129A mutant, which lacks the general acid/base residue Asp-129, an alternative reaction mechanism is proposed. The calculated activation barriers are in all cases in good agreement with experimental reaction rates. The present results together with earlier computational and experimental work now provide a detailed picture of the complete reaction mechanism in many PTPs. The key role played by the structurally invariant signature motif in stabilizing a double negative charge is reflected by its control of the energetics of both transition states and the reaction intermediate.     

Mechanistic differences among retaining disaccharide phosphorylases: insights from kinetic analysis of active site mutants of sucrose phosphorylase and alpha,alpha-trehalose phosphorylase

Sucrose phosphorylase utilizes a glycoside hydrolase-like double displacement mechanism to convert its disaccharide substrate and phosphate into alpha-d-glucose 1-phosphate and fructose. Site-directed mutagenesis was employed to characterize the proposed roles of Asp(196) and Glu(237) as catalytic nucleophile and acid-base, respectively, in the reaction of sucrose phosphorylase from Leuconostoc mesenteroides. The side chain of Asp(295) is suggested to facilitate the catalytic steps of glucosylation and deglucosylation of Asp(196) through a strong hydrogen bond (23 kJ/mol) with the 2-hydroxyl of the glucosyl oxocarbenium ion-like species believed to be formed in the transition states flanking the beta-glucosyl enzyme intermediate. An assortment of biochemical techniques used to examine the mechanism of alpha-retaining glucosyl transfer by Schizophyllum commune alpha,alpha-trehalose phosphorylase failed to provide evidence in support of a similar two-step catalytic reaction via a covalent intermediate. Mutagenesis studies suggested a putative active-site structure for this trehalose phosphorylase that is typical of retaining glycosyltransferases of fold family GT-B and markedly different from that of sucrose phosphorylase. While ambiguity remains regarding the chemical mechanism by which the trehalose phosphorylase functions, the two disaccharide phosphorylases have evolved strikingly different reaction coordinates to achieve catalytic efficiency and stereochemical control in their highly analogous substrate transformations.     

Reaction of alkylcobalamins with thiols

Carbon-13 NMR spectroscopy and phosphorus-31 NMR spectroscopy have been used to study the reaction of several alkylcobalamins with 2-mercaptoethanol. At alkaline pH, when the thiol is deprotonated, the alkyl-transfer reactions involve a nucleophilic attack of the thiolate anion on the Co-methylene carbon of the cobalamins, yielding alkyl thioethers and cob(II)alamin. In these nucleophilic displacement reactions cob(I)alamin is presumably formed as an intermediate. The higher alkylcobalamins react more slowly than methylcobalamin. The lower reactivity of ethyl- and propylcobalamin is probably the basis of the inhibition of the corrinoid-dependent methyl-transfer systems by propyl iodide. The transfer of the upper nucleoside ligand of adenosylcobalamin to 2-mercaptoethanol is a very slow process; S-adenosyl-mercaptoethanol and cob(II)alamin are the final products of the reaction. The dealkylation of (carboxymethyl)cobalamin is a much more facile reaction. At alkaline pH S-(carboxymethyl)mercaptoethanol and cob(II)alamin are produced, while at pH values below 8 the carbon-cobalt bond is cleaved reductively to acetate and cob(II)alamin. The reductive cleavage of the carbon-cobalt bond of (carboxymethyl)cobalamin by 2-mercaptoethanol is extremely fast when the cobalamin is in the "base-off" form. Because we have been unable to detect trans coordination of 2-mercaptoethanol, we favor a mechanism that involves a hydride attack on the Co-methylene carbon of (carboxymethyl)cobalamin rather than a trans attack of the thiol on the cobalt atom.     

The specificity of the synthetic reaction of two yeast alpha-glucosidases.

The specificity of the hydrolytic reaction has been compared to that of the synthetic reaction for maltase and isomaltase (alpha-methyl-D-glucosidase) from Saccharomyces oviformis. Maltase which hydrolyzes the alpha-1,4-disaccharide, maltose, and the alpha-1,6-disaccharide, isomaltose, catalyzes the formation of both maltose and isomaltose from free glucose. Isomaltase, which hydrolyzes isomaltose but not maltose, catalyzes the formation only of isomaltose from glucose. Both enzymes hydrolyze p-nitrophenyl-alpha-D-glucoside releasing the alpha-anomer of glucose. The enzymes utilize the alpha-anomer but not the beta-anomer for the synthesis of the disaccharides. These results are consistent with the double displacement mechanism for glycosidases and with the proposal that the glucosyl-enzyme complex is an intermediate in the reaction. The competitive inhibition by D-glucose is independent of its anomeric form for both enzymes.     

The reaction catalyzed by tetrachlorohydroquinone dehalogenase does not involve nucleophilic aromatic substitution.

Tetrachlorohydroquinone dehalogenase catalyzes the reductive dehalogenation of tetrachlorohydroquinone and trichlorohydroquinone during the biodegradation of the xenobiotic compound pentachlorophenol by Sphingobium chlorophenolicum. The mechanism of this transformation is of interest because it is unusual and difficult, and because aerobic microorganisms rarely catalyze reductive dehalogenation reactions. Tetrachlorohydroquinone dehalogenase is a member of the glutathione S-transferase superfamily. Many enzymes in this superfamily are capable of catalyzing nucleophilic aromatic substitution reactions. On the basis of this precedent, we have considered a mechanism for tetrachlorohydroquinone dehalogenase that involves a nucleophilic aromatic substitution reaction, either via an S(N)Ar mechanism or an S(RN)1-like mechanism, in the initial part of the reaction. Mechanistic studies were carried out with the wild type enzyme and with the C13S mutant enzyme, which catalyzes only the initial steps in the reaction. Three findings eliminate the possibility of a nucleophilic aromatic substitution reaction. First, the product of such a reaction, 2,3,5-trichloro-6-S-glutathionylhydroquinone, is not a kinetically competent intermediate. Second, the enzyme can carry out the reaction when the substrate is deprotonated at the active site. Nucleophilic aromatic substitution should not be possible when the substrate is negatively charged. Third, substantial normal solvent kinetic isotope effects on k(cat) and k(cat)/K(M,TriCHQ) are observed. Nonenzymatic and enzymatic nucleophilic S(N)Ar reactions typically show inverse solvent kinetic isotope effects.     

Biological reactions at phosphorus: VII. The nature of metaphosphate ion,PO3−, as a reaction intermediate (1)

In order to improve our understanding of biological phosphorylations by “high-energy” compounds such as ATP, the hypothesis of metaphosphate ion as an intermediate in certain phosphorylation reactions has been critically examined. We have studied the rates and product composition for the solvolysis of the neutral form of N,N-dimethylphosphoroguanidinate (DMPG) at 30.5°C in various water-alcohol mixtures. The rates of solvolysis were found to decrease as the mole percent of the alcohol increased, but no systematic relationship with dielectric constant or Grunwald-Winstein y values was evident. A 1 : 1 correspondence between the percentage alkyl phosphate produced and the mole percent alcohol present was found with methanol, ethanol, and low concentrations of 2-propanol. At higher concentrations of 2-propanol, the product ratio favors water as nucleophile probably due to selective solvation of the metaphosphate precursor by water. These results indicate that metaphosphate mechanisms have a variable amount of nucleophilic participation. Although the reaction of phosphoroguanidines appears to involve metaphosphate ion as a free intermediate, analysis of results in the literature indicate that less reactive metaphosphate precursors react with nucleophilic participation. Extrapolation of these results to biological phosphorylations leads to the conclusion that nucleophilic participation may be an important feature of enzymic transition states due to the favorable orientation of nucleophile and incipient metaphosphate at enzymic active sites.     

Stepwise Catalytic Mechanism via Short-Lived Intermediate Inferred from Combined QM/MM MERP and PES Calculations on Retaining Glycosyltransferase ppGalNAcT2

The glycosylation of cell surface proteins plays a crucial role in a multitude of biological processes, such as cell adhesion and recognition. To understand the process of protein glycosylation, the reaction mechanisms of the participating enzymes need to be known. However, the reaction mechanism of retaining glycosyltransferases has not yet been sufficiently explained. Here we investigated the catalytic mechanism of human isoform 2 of the retaining glycosyltransferase polypeptide UDP-GalNAc transferase by coupling two different QM/MM-based approaches, namely a potential energy surface scan in two distance difference dimensions and a minimum energy reaction path optimisation using the Nudged Elastic Band method. Potential energy scan studies often suffer from inadequate sampling of reactive processes due to a predefined scan coordinate system. At the same time, path optimisation methods enable the sampling of a virtually unlimited number of dimensions, but their results cannot be unambiguously interpreted without knowledge of the potential energy surface. By combining these methods, we have been able to eliminate the most significant sources of potential errors inherent to each of these approaches. The structural model is based on the crystal structure of human isoform 2. In the QM/MM method, the QM region consists of 275 atoms, the remaining 5776 atoms were in the MM region. We found that ppGalNAcT2 catalyzes a same-face nucleophilic substitution with internal return (S_Ni). The optimized transition state for the reaction is 13.8 kcal/mol higher in energy than the reactant while the energy of the product complex is 6.7 kcal/mol lower. During the process of nucleophilic attack, a proton is synchronously transferred to the leaving phosphate. The presence of a short-lived metastable oxocarbenium intermediate is likely, as indicated by the reaction energy profiles obtained using high-level density functionals.     

Mechanistic analysis of the unusual redox-elimination sequence employed by Thermotoga maritima BglT: a 6-phospho-beta-glucosidase from glycoside hydrolase family 4

"Classical" glycosidases utilize either direct or double-displacement mechanisms involving oxocarbenium ion-like transition states to catalyze the hydrolysis of glycosidic bonds. By contrast, the mechanism of the glycosidases in glycoside hydrolase family 4 has been recently proposed to involve NAD+-mediated redox steps along with alpha,beta-elimination and addition steps via anionic intermediates. Support for this mechanism in BglT, a 6-phospho-beta-glucosidase in family 4, has been provided through mechanistic and X-ray crystallographic analyses [Yip, V. L.Y., et al. (2004) J. Am. Chem. Soc. 126, 8354-8355] in which primary deuterium kinetic isotope effects for the hydride abstraction at C3 and for the alpha-proton abstraction at C2 indicate that these two steps are both partially rate-limiting. Current data reveal that there is no secondary deuterium kinetic isotope effect associated with the rehybridization of the C1 sp3 center to a sp2 center. Furthermore, a flat linear free energy relationship was established with a series of aryl 6-phospho-beta-D-glucosides of varying leaving group abilities. Taken together, these data indicate that cleavage of the C1-O1 linkage does not occur during a rate-limiting step. Since the deprotonation at C2 is slow and partially rate-limiting while the departure of the leaving group is not, a stepwise E1(cb)-type mechanism rather than an E1 or a concerted E2-syn mechanism is proposed. Direct evidence for the role of NAD+ was obtained by reduction in situ using NaBH4 leading to an inactive enzyme that could be reactivated by the addition of excess NAD+. This was accompanied by the expected UV-vis spectrophotometric changes.     

Effects of Cations and Other Medium Components on the Zona-induced Acrosome Reaction of Hamster Spermatozoa

Although the acrosome reaction in lively motile hamster spermatozoa can occur independently of the egg or its investments ("spontaneous" acrosome reaction), it appears to be the egg investments, particularly the zona pellucida, that induces the acrosome reaction in fertilizing spermatozoa of many mammalian species. The latter is referred to as "zona-induced" acrosome reaction. Experiments were conducted to determine if the zona-induced acrosome reaction has different ion requirements from the spontaneous reaction. Like the spontaneous acrosome reaction, the zona-induced acrosome reaction required extracellular Na⁺, K⁺ and Ca²⁺. The absence of Cl⁻ and albumin in the medium inhibited the reaction. The zona-induced acrosome reaction could occur in a HCO₃⁻-free medium, but far less efficiently than in medium containing this ion. Proteinase inhibitors, benzamidine and TLCK, inhibited the zona-induced acrosome reaction. These results suggest that the chemical reactions involved in the spontaneous and zona-induced acrosome reactions are similar although the reaction-triggering mechanism is probably different.     

Reviving a dead enzyme: cytosine deaminations promoted by an inactive DNA methyltransferase and an S-adenosylmethionine analogue

The enzymes that transfer a methyl group to C5 of cytosine within specific sequences (C5 Mtases) deaminate the target cytosine to uracil if the methyl donor S-adenosylmethionine (SAM) is omitted from the reaction. Recently, it was shown that cytosine deamination caused by C5 Mtases M.HpaII, M.SssI and M.MspI is enhanced in the presence of several analogues of SAM, and a mechanism for this analogue-promoted deamination was proposed. According to this mechanism, the analogues protonate C5 of the target cytosine, creating a dihydrocytosine intermediate that is susceptible to deamination. We show here that one of these analogues, 5'-aminoadenosine (AA), enhances cytosine deamination by the Mtase M. EcoRII, but it does so without enhancing protonation of C5. Further, we show that uracil is an intermediate in the mutational pathway and propose an alternate mechanism for the analogue-promoted deamination. The new mechanism involves a facilitated water attack at C4 but does not require attack at C6 by the enzyme. The latter feature of the mechanism was tested by using M.EcoRII mutants defective in the nucleophilic attack at C6 in the deamination assay. We find that although these proteins are defective in methyl transfer and cytosine deamination, they cause cytosine deaminations in the presence of AA in the reaction. Our results point to a possible connection between the catalytic mechanism of C5 Mtases and of enzymes that transfer methyl groups to N(4) of cytosine. Further, they provide an unusual example where a coenzyme activates an otherwise "dead" enzyme to perform catalysis by a new reaction pathway.     

ATP-citrate lyase from rat liver. Characterisation of the citryl-enzyme complexes.

The mechanism of ATP-citrate lyase has been proposed to involve a citryl-enzyme intermediate. When the enzyme is incubated with its substrates ATP and [14C]citrate, but in the absence of the final acceptor, two distinct types of citrate-containing complex can be isolated. At early time points, a highly unstable complex can be isolated by gel filtration which has a half-life of 36 s at 25 degrees C. This complex reacts rapidly with CoA, but cannot be acid-precipitated; behaviour consistent with its identification as enzyme-citryl phosphate. However, ATP-citrate lyase is also capable of undergoing a slow time-dependent covalent incorporation of radiolabel from [14C]citrate. This modification is acid-stable, non-specific, and cannot be reversed by the addition of CoA. When cytochrome is included in the reaction mixture as a heterologous acceptor, it is also citrylated. These reactions require that when ATP-citrate lyase is incubated with all its substrates except for CoA, a freely diffusible citrylating species is generated within the active site. This evidence suggests that there is no requirement for the mechanism of ATP-citrate lyase to proceed via a covalent citryl-enzyme intermediate. By analogy with succinyl-CoA synthetase, an enzyme which has a high degree of sequence similarity with ATP-citrate lyase, a simple mechanism is proposed for the enzyme in which citryl-CoA is produced by direct nucleophilic attack on citryl phosphate.