Characterization of the amino acids involved in substrate specificity of nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase from Streptococcus mutans

In order to address the molecular basis of the specificity of aldehyde dehydrogenase for aldehyde substrates, enzymatic characterization of the glyceraldehyde 3-phosphate (G3P) binding site of non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN) from Streptococcus mutans has been undertaken. In this work, residues Arg-124, Tyr-170, Arg-301, and Arg-459 were changed by site-directed mutagenesis and the catalytic properties of GAPN mutants investigated. Changing Tyr-170 into phenylalanine induces no major effect on k(cat) and K(m) for d-G3P in both acylation and deacylation steps. Substitutions of Arg-124 and Arg-301 by leucine and Arg-459 by isoleucine led to distinct effects on K(m), on k(cat), or on both. The rate-limiting step of the R124L GAPN remains deacylation. Pre-steady-state analysis and substrate isotope measurements show that hydride transfer remains rate-determining in acylation. Only the apparent affinity for d-G3P is decreased in both acylation and deacylation steps. Substitution of Arg-459 by isoleucine leads to a drastic effect on the catalytic efficiency by a factor of 10(5). With this R459L GAPN, the rate-limiting step is prior to hydride transfer, and the K(m) of d-G3P is increased by at least 2 orders of magnitude. Binding of NADP leads to a time-dependent formation of a charge transfer transition at 333 nm between the pyridinium ring of NADP and the thiolate of Cys-302, which is not observed with the holo-wild type. Accessibility of Cys-302 is shown to be strongly decreased within the holostructure. The substitution of Arg-301 by leucine leads to an even more drastic effect with a change of the rate-limiting step similar to that observed for R459I GAPN. Taking into account the three-dimensional structure of GAPN from S. mutans and the data of the present study, it is proposed that 1) Tyr-170 is not essential for the catalytic event, 2) Arg-124 is only involved in stabilizing d-G3P binding via an interaction with the C-3 phosphate, and 3) Arg-301 and Arg-459 participate not only in d-G3P binding via interaction with C-3 phosphate but also in positioning efficiently d-G3P relative to Cys-302 within the ternary complex GAPN.NADP.d-G3P.     

Mechanistic characterization of the MSDH (methylmalonate semialdehyde dehydrogenase) from Bacillus subtilis

Homotetrameric MSDH (methylmalonate semialdehyde dehydrogenase) from Bacillus subtilis catalyses the NAD-dependent oxidation of MMSA (methylmalonate semialdehyde) and MSA (malonate semialdehyde) into PPCoA (propionyl-CoA) and acetyl-CoA respectively via a two-step mechanism. In the present study, a detailed mechanistic characterization of the MSDH-catalysed reaction has been carried out. The results suggest that NAD binding elicits a structural imprinting of the apoenzyme, which explains the marked lag-phase observed in the activity assay. The enzyme also exhibits a half-of-the-sites reactivity, with two subunits being active per tetramer. This result correlates well with the presence of two populations of catalytic Cys302 in both the apo- and holo-enzymes. Binding of NAD causes a decrease in reactivity of the two Cys302 residues belonging to the two active subunits and a pKapp shift from approx. 8.8 to 8.0. A study of the rate of acylation as a function of pH revealed a decrease in the pKapp of the two active Cys302 residues to approx. 5.5. Taken to-gether, these results support a sequential Cys302 activation process with a pKapp shift from approx. 8.8 in the apo-form to 8.0 in the binary complex and finally to approx. 5.5 in the ternary complex. The rate-limiting step is associated with the b-decarboxylation process which occurs on the thioacylenzyme intermediate after NADH release and before transthioesterification. These data also indicate that bicarbonate, the formation of which is enzyme-catalysed, is the end-product of the reaction.     

Adenine Binding Mode Is a Key Factor in Triggering the Early Release of NADH in Coenzyme A-dependent Methylmalonate Semialdehyde Dehydrogenase

Structural dynamics associated with cofactor binding have been shown to play key roles in the catalytic mechanism of hydrolytic NAD(P)-dependent aldehyde dehydrogenases (ALDH). By contrast, no information is available for their CoA-dependent counterparts. We present here the first crystal structure of a CoA-dependent ALDH. The structure of the methylmalonate semialdehyde dehydrogenase (MSDH) from Bacillus subtilis in binary complex with NAD(+) shows that, in contrast to what is observed for hydrolytic ALDHs, the nicotinamide ring is well defined in the electron density due to direct and H(2)O-mediated hydrogen bonds with the carboxamide. The structure also reveals that a conformational isomerization of the NMNH is possible in MSDH, as shown for hydrolytic ALDHs. Finally, the adenine ring is substantially more solvent-exposed, a result that could be explained by the presence of a Val residue at position 229 in helix α(F) that reduces the depth of the binding pocket and the absence of Gly-225 at the N-terminal end of helix α(F). Substitution of glycine for Val-229 and/or insertion of a glycine residue at position 225 resulted in a significant decrease of the rate constant associated with the dissociation of NADH from the NADH/thioacylenzyme complex, thus demonstrating that the weaker stabilization of the adenine ring is a key factor in triggering the early NADH release in the MSDH-catalyzed reaction. This study provides for the first time structural insights into the mechanism whereby the cofactor binding mode is responsible at least in part for the different kinetic behaviors of the hydrolytic and CoA-dependent ALDHs.     

Structural Basis for a Bispecific NADP+ and CoA Binding Site in an Archaeal Malonyl-Coenzyme A Reductase

Autotrophic members of the Sulfolobales (crenarchaeota) use the 3-hydroxypropionate/4-hydroxybutyrate cycle to assimilate CO₂ into cell material. The product of the initial acetyl-CoA carboxylation with CO₂, malonyl-CoA, is further reduced to malonic semialdehyde by an NADPH-dependent malonyl-CoA reductase (MCR); the enzyme also catalyzes the reduction of succinyl-CoA to succinic semialdehyde onwards in the cycle. Here, we present the crystal structure of Sulfolobus tokodaii malonyl-CoA reductase in the substrate-free state and in complex with NADP⁺ and CoA. Structural analysis revealed an unexpected reaction cycle in which NADP⁺ and CoA successively occupy identical binding sites. Both coenzymes are pressed into an S-shaped, nearly superimposable structure imposed by a fixed and preformed binding site. The template-governed cofactor shaping implicates the same binding site for the 3′- and 2′-ribose phosphate group of CoA and NADP⁺, respectively, but a different one for the common ADP part: the β-phosphate of CoA aligns with the α-phosphate of NADP⁺. Evolution from an NADP⁺ to a bispecific NADP⁺ and CoA binding site involves many amino acid exchanges within a complex process by which constraints of the CoA structure also influence NADP⁺ binding. Based on the paralogous aspartate-β-semialdehyde dehydrogenase structurally characterized with a covalent Cys-aspartyl adduct, a malonyl/succinyl group can be reliably modeled into MCR and discussed regarding its binding mode, the malonyl/succinyl specificity, and the catalyzed reaction. The modified polypeptide surrounding around the absent ammonium group in malonate/succinate compared with aspartate provides the structural basis for engineering a methylmalonyl-CoA reductase applied for biotechnical polyester building block synthesis.     

Catalysis by dienelactone hydrolase: A variation on the protease mechanism

Dienelactone hydrolase (DLH), an enzyme from the β-ketoadipate pathway, catalyzes the hydrolysis of dienelactone to maleylacetate. Our inhibitor binding studies suggest that its substrate, dienelactone, is held in the active site by hydrophobic interactions around the lactone ring and by the ion pairs between its carboxylate and Arg-81 and Arg-206. Like the cysteine/serine proteases, DLH has a catalytic triad (Cys-123, His-202, Asp-171) and its mechanism probably involves the formation of covalently bound acyl intermediate via a tetrahedral intermediate. Unlike the proteases, DLH seems to protonate the incipient leaving group only after the collapse of the first tetrahedral intermediate, rendering DLH incapable of hydrolyzing amide analogues of its ester substrate. In addition, the triad His probably does not protonate the leaving group (enolate) or deprotonate the water for deacylation; rather, the enolate anion abstracts a proton from water and, in doing so, supplies the hydroxyl for deacylation. © 1993 Wiley-Liss, Inc.     

Exploring structural motifs necessary for substrate binding in the active site of Escherichia coli pantothenate kinase

The coenzyme A (CoA) biosynthetic enzymes have been used to produce various CoA analogues, including mechanistic probes of CoA-dependent enzymes such as those involved in fatty acid biosynthesis. These enzymes are also important for the activation of the pantothenamide class of antibacterial agents, and of a recently reported family of antibiotic resistance inhibitors. Herein we report a study on the selectivity of pantothenate kinase, the first and rate limiting step of CoA biosynthesis. A robust synthetic route was developed to allow rapid access to a small library of pantothenate analogs diversified at the β-alanine moiety, the carboxylate or the geminal dimethyl group. All derivatives were tested as substrates of Escherichia coli pantothenate kinase (EcPanK). Four derivatives, all N-aromatic pantothenamides, proved to be equivalent to the benchmark N-pentylpantothenamide (N5-pan) as substrates of EcPanK, while two others, also with N-aromatic groups, were some of the best substrates reported for this enzyme. This collection of data provides insight for the future design of PanK substrates in the production of useful CoA analogues.     

The importance of the amide bond nearest the thiol group in enzymatic reactions of coenzyme A

Analogues of coenzyme A (CoA) and of CoA thioesters have been prepared in which the amide bond nearest the thiol group has been modified. An analogue of acetyl-CoA in which this amide bond is replaced with an ester linkage was a good substrate for the enzymes carnitine acetyltransferase, chloramphenicol acetyltransferase, and citrate synthase, with K(m) values 2- to 8-fold higher than those of acetyl-CoA and V(max) values from 14 to >80% those of the natural substrate. An analogue in which an extra methylene group was inserted between the amide bond and the thiol group showed less than 4-fold diminished binding to the three enzymes but exhibited less than 1% activity relative to acetyl-CoA with carnitine acetyltransferase and no measurable activity with the other two enzymes. Analogues of several CoA thioesters in which the amide bond was replaced with a hemithioacetal linkage exhibited no measurable activity with the appropriate enzymes. The results indicate that some aspects of the amide bond and proper distance between this amide and the thiol/thioester moiety are critical for activity of CoA ester-utilizing enzymes.     

Chemical mechanism and substrate binding sites of NADP-dependent aldehyde dehydrogenase from Streptococcus mutans

Non-phosphorylating glyceraldehyde 3-phosphate dehydrogenase from Streptococcus mutans (GAPN) belongs to the aldehyde dehydrogenase (ALDH) family, which catalyzes the irreversible oxidation of a wide variety of aldehydes into acidic compounds via a two-step mechanism: first, the acylation step involves the formation of a covalent ternary complex ALDH-cofactor-substrate, followed by the oxidoreduction process which yields a thioacyl intermediate and reduced cofactor and second, the rate-limiting deacylation step. Structural and molecular factors involved in the chemical mechanism of GAPN have recently been examined. Specifically, evidence was put forward for the chemical activation of catalytic Cys-302 upon cofactor binding to the enzyme, through a local conformational rearrangement involving the cofactor and Glu-268. In addition, the invariant residue Glu-268 was shown to play an essential role in the activation of the water molecule in the deacylation step. For E268A/Q mutant GAPNs, nucleophilic compounds like hydrazine and hydroxylamine were shown to bind and act as substrates in this step. Further studies were focused at understanding the factors responsible for the stabilization and chemical activation of the covalent intermediates, using X-ray crystallography, site-directed mutagenesis, kinetic and physico-chemical approaches. The results support the involvement of an oxyanion site including the side-chain of Asn-169. Finally, given the strict substrate-specificity of GAPN compared to other ALDHs with wide substrate specificity, one has also initiated the characterization of the G3P binding properties of GAPN. These results will be presented and discussed from the point of view of the evolution of the catalytic mechanisms of ALDH.     

Chemical mechanism and substrate binding sites of NADP-dependent aldehyde dehydrogenase from Streptococcus mutans

Non-phosphorylating glyceraldehyde 3-phosphate dehydrogenase from Streptococcus mutans (GAPN) belongs to the aldehyde dehydrogenase (ALDH) family, which catalyzes the irreversible oxidation of a wide variety of aldehydes into acidic compounds via a two-step mechanism: first, the acylation step involves the formation of a covalent ternary complex ALDH-cofactor-substrate, followed by the oxidoreduction process which yields a thioacyl intermediate and reduced cofactor and second, the rate-limiting deacylation step. Structural and molecular factors involved in the chemical mechanism of GAPN have recently been examined. Specifically, evidence was put forward for the chemical activation of catalytic Cys-302 upon cofactor binding to the enzyme, through a local conformational rearrangement involving the cofactor and Glu-268. In addition, the invariant residue Glu-268 was shown to play an essential role in the activation of the water molecule in the deacylation step. For E268A/Q mutant GAPNs, nucleophilic compounds like hydrazine and hydroxylamine were shown to bind and act as substrates in this step. Further studies were focused at understanding the factors responsible for the stabilization and chemical activation of the covalent intermediates, using X-ray crystallography, site-directed mutagenesis, kinetic and physico-chemical approaches. The results support the involvement of an oxyanion site including the side-chain of Asn-169. Finally, given the strict substrate-specificity of GAPN compared to other ALDHs with wide substrate specificity, one has also initiated the characterization of the G3P binding properties of GAPN. These results will be presented and discussed from the point of view of the evolution of the catalytic mechanisms of ALDH.     

Studies on the mechanism of ring hydrolysis in phenylacetate degradation: a metabolic branching point

The widespread, long sought-after bacterial aerobic phenylalanine/phenylacetate catabolic pathway has recently been elucidated. It proceeds via coenzyme A (CoA) thioesters and involves the epoxidation of the aromatic ring of phenylacetyl-CoA, subsequent isomerization to an uncommon seven-membered C-O-heterocycle (oxepin-CoA), and non-oxygenolytic ring cleavage. Here we characterize the hydrolytic oxepin-CoA ring cleavage catalyzed by the bifunctional fusion protein PaaZ. The enzyme consists of a C-terminal (R)-specific enoyl-CoA hydratase domain (formerly MaoC) that cleaves the ring and produces a highly reactive aldehyde and an N-terminal NADP(+)-dependent aldehyde dehydrogenase domain that oxidizes the aldehyde to 3-oxo-5,6-dehydrosuberyl-CoA. In many phenylacetate-utilizing bacteria, the genes for the pathway exist in a cluster that contains an NAD(+)-dependent aldehyde dehydrogenase in place of PaaZ, whereas the aldehyde-producing hydratase is encoded outside of the cluster. If not oxidized immediately, the reactive aldehyde condenses intramolecularly to a stable cyclic derivative that is largely prevented by PaaZ fusion in vivo. Interestingly, the derivative likely serves as the starting material for the synthesis of antibiotics (e.g. tropodithietic acid) and other tropone/tropolone related compounds as well as for ω-cycloheptyl fatty acids. Apparently, bacteria made a virtue out of the necessity of disposing the dead-end product with ring hydrolysis as a metabolic branching point.     

The first crystal structure of a thioacylenzyme intermediate in the ALDH family: new coenzyme conformation and relevance to catalysis

Crystal structures of several members of the nonphosphorylating CoA-independent aldehyde dehydrogenase (ALDH) family have shown that the peculiar binding mode of the cofactor to the Rossmann fold results in a conformational flexibility for the nicotinamide moiety of the cofactor. This has been hypothesized to constitute an essential feature of the catalytic mechanism because the conformation of the cofactor required for the acylation step is not appropriate for the deacylation step. In the present study, the structure of a reaction intermediate of the E268A-glyceraldehyde 3-phosphate dehydrogenase (GAPN) from Streptococcus mutans, obtained by soaking the crystals of the enzyme/NADP complex with the natural substrate, is reported. The substrate is bound covalently in the four monomers and presents the geometric characteristics expected for a thioacylenzyme intermediate. Control experiments assessed that reduction of the coenzyme has occurred within the crystal. The structure reveals that reduction of the cofactor upon acylation leads to an extensive motion of the nicotinamide moiety with a flip of the reduced pyridinium ring away from the active site without significant changes of the protein structure. This event positions the reduced nicotinamide moiety in a pocket that likely constitutes the exit door for NADPH. Arguments are provided that the structure reported here constitutes a reasonable picture of the first thioacylenzyme intermediate characterized thus far in the ALDH family and that the position of the reduced nicotinamide moiety observed in GAPN is the one suitable for the deacylation step within all of the nonphosphorylating CoA-independent ALDH family.     

Cancer-associated isocitrate dehydrogenase mutations inactivate NADPH-dependent reductive carboxylation

Isocitrate dehydrogenase (IDH) is a reversible enzyme that catalyzes the NADP(+)-dependent oxidative decarboxylation of isocitrate (ICT) to α-ketoglutarate (αKG) and the NADPH/CO(2)-dependent reductive carboxylation of αKG to ICT. Reductive carboxylation by IDH1 was potently inhibited by NADP(+) and, to a lesser extent, by ICT. IDH1 and IDH2 with cancer-associated mutations at the active site arginines were unable to carry out the reductive carboxylation of αKG. These mutants were also defective in ICT decarboxylation and converted αKG to 2-hydroxyglutarate using NADPH. These mutant proteins were thus defective in both of the normal reactions of IDH. Biochemical analysis of heterodimers between wild-type and mutant IDH1 subunits showed that the mutant subunit did not inactivate reductive carboxylation by the wild-type subunit. Cells expressing the mutant IDH are thus deficient in their capacity for reductive carboxylation and may be compromised in their ability to produce acetyl-CoA under hypoxia or when mitochondrial function is otherwise impaired.     

Structure and mechanism of human UDP-glucose 6-dehydrogenase

Elevated production of the matrix glycosaminoglycan hyaluronan is strongly implicated in epithelial tumor progression. Inhibition of synthesis of the hyaluronan precursor UDP-glucuronic acid (UDP-GlcUA) therefore presents an emerging target for cancer therapy. Human UDP-glucose 6-dehydrogenase (hUGDH) catalyzes, in two NAD(+)-dependent steps without release of intermediate aldehyde, the biosynthetic oxidation of UDP-glucose (UDP-Glc) to UDP-GlcUA. Here, we present a structural characterization of the hUGDH reaction coordinate using crystal structures of the apoenzyme and ternary complexes of the enzyme bound with UDP-Glc/NADH and UDP-GlcUA/NAD(+). The quaternary structure of hUGDH is a disc-shaped trimer of homodimers whose subunits consist of two discrete α/β domains with the active site located in the interdomain cleft. Ternary complex formation is accompanied by rigid-body and restrained movement of the N-terminal NAD(+) binding domain, sequestering substrate and coenzyme in their reactive positions through interdomain closure. By alternating between conformations in and out of the active site during domain motion, Tyr(14), Glu(161), and Glu(165) participate in control of coenzyme binding and release during 2-fold oxidation. The proposed mechanism of hUGDH involves formation and breakdown of thiohemiacetal and thioester intermediates whereby Cys(276) functions as the catalytic nucleophile. Stopped-flow kinetic data capture the essential deprotonation of Cys(276) in the course of the first oxidation step, allowing the thiolate side chain to act as a trap of the incipient aldehyde. Because thiohemiacetal intermediate accumulates at steady state under physiological reaction conditions, hUGDH inhibition might best explore ligand binding to the NAD(+) binding domain.     

Chemical modification of the functional arginine residues of carbon monoxide dehydrogenase from Clostridium thermoaceticum

Carbon monoxide dehydrogenase (CODH) is the key enzyme of autotrophic growth with CO or CO2 and H2 by the acetyl-CoA pathway. The enzyme from Clostridium thermoaceticum catalyzes the formation of acetyl-CoA from the methyl, carbonyl, and CoA groups and has separate binding sites for these moieties. In this study, we have determined the role of arginine residues in binding of CoA by CODH. Phenylglyoxal, an arginine-specific reagent, inactivated CODH, and CoA afforded about 80-85% protection against this inactivation. The other ligands, such as the carbonyl and the methyl groups, gave no protection. By circular dichroism, it was shown that the loss of activity is not due to extensive structural changes in CODH. Earlier, we showed that tryptophan residues are located at the CoA binding site of CODH [Shanmugasundaram, T., Kumar, G. K., & Wood, H. G. (1988) Biochemistry 27, 6499-6503]. A comparison of the fluorescence spectra of the native and phenylglyoxal-modified enzymes indicates that the reactive arginine residues appear to be located close to fluorescing tryptophans. Fluorescence spectral studies with CoA analogues or its components showed that CoA interacts with the tryptophan(s) of CODH through its adenine moiety. In addition, evidence is presented that the arginines interact with the pyrophosphate moiety of CoA.     

Crystal structures of the carboxyl terminal domain of rat 10-formyltetrahydrofolate dehydrogenase: implications for the catalytic mechanism of aldehyde dehydrogenases

10-Formyltetrahydrofolate dehydrogenase (FDH) catalyzes an NADP+-dependent dehydrogenase reaction resulting in conversion of 10-formyltetrahydrofolate to tetrahydrofolate and CO2. This reaction is a result of the concerted action of two catalytic domains of FDH, the amino-terminal hydrolase domain and the carboxyl-terminal aldehyde dehydrogenase domain. In addition to participation in the overall FDH mechanism, the C-terminal domain is capable of NADP+-dependent oxidation of short chain aldehydes to their corresponding acids. We have determined the crystal structure of the C-terminal domain of FDH and its complexes with oxidized and reduced forms of NADP. Compared to other members of the ALDH family, FDH demonstrates a new mode of binding of the 2'-phosphate group of NADP via a water-mediated contact with Gln600 that may contribute to the specificity of the enzyme for NADP over NAD. The structures also suggest how Glu673 can act as a general base in both acylation and deacylation steps of the reaction. In the apo structure, the general base Glu673 is positioned optimally for proton abstraction from the sulfur atom of Cys707. Upon binding of NADP+, the side chain of Glu673 is displaced from the active site by the nicotinamide ring and contacts a chain of highly ordered water molecules that may represent a pathway for translocation of the abstracted proton from Glu673 to the solvent. When reduced, the nicotinamide ring of NADP is displaced from the active site, restoring the contact between Cys707 and Glu673 and allowing the latter to activate the hydrolytic water molecule in deacylation.     

Inhibition of cathepsin G by 2-amino-3,1-benzoxazin-4-ones: kinetic investigations and docking studies

A series of benzoxazinones was used to investigate the interaction of human cathepsin G with acyl-enzyme inhibitors. With respect to the primary specificity of cathepsin G, inhibitors with hydrophobic or basic residues at position 2 were included in the study. Parameters of the enzyme acylation and deacylation were determined by slow-binding kinetics in the presence of a chromogenic substrate. For selected inhibitors, the time course of the enzyme-catalyzed conversion of the inhibitors was followed. This approach was suitable to elucidate a rate-determining deacylation step. Docking simulations of the noncovalent enzyme-inhibitor complexes were performed and several clusters were analyzed for each inhibitor. The amino acids of the active site that participate in the binding of the inhibitors were determined. The arrangements in several clusters of an inhibitor were not uniform with respect to the orientation by which the inhibitor was bound in the S(1) pocket. Docking of the basic piperazino derivatives 6 and 10 indicated an interaction with Glu 226 at the bottom of the S(1) specificity pocket. The (N-methyl)benzylamino derivative 1 showed the strongest acylation rate (k(on)=1200 M(-1) s(-1)), which was attributed to a high extent of pseudo-productive orientations of the noncovalent preassociation complex.     

Engineered nonphosphorylating glyceraldehyde 3-phosphate dehydrogenase at position 268 binds hydroxylamine and hydrazine as acyl acceptors.

Nonphosphorylating nicotinamide adenine dinucleotide (phosphate)-dependent aldehyde dehydrogenases (ALDHs) catalyze the oxidation of aldehydes into either nonactivated acids or CoA-activated acids. The NADP-dependent nonphosphorylating glyceraldehyde 3-phosphate dehydrogenase (GAPN) belongs to the first subclass. It catalyzes the irreversible oxidation of glyceraldehyde 3-phosphate into 3-phosphoglycerate via a two step mechanism in which deacylation is rate-limiting. Recent studies on GAPN from Streptococcus mutans have shown that residue Glu268 plays an essential role only in the deacylation step [Marchal, S., Rahuel-Clermont, S. & Branlant, G. (2000) Biochemistry 39, 3327-3335]. The substitution of Glu268 by alanine or glutamine leads to mutants in which the attacking water molecule involved in the hydrolytic process is poorly activated. Activity can be restored by the presence of hydroxylamine and hydrazine. Neutral and protonated forms of both nucleophiles are recognized by the deacylating subsite of both mutants. pH rate profiles of deacylation show pK(a) values of 6.3 and 8.1 with hydroxylamine and hydrazine, respectively, which are those of the nucleophiles in solution. The increase in enzymatic rate is probably due to a high local concentration and not to a change of the chemical reactivity of both nucleophiles upon their binding within the active site of both mutants. The deacylation subsite of the wild-type also binds hydroxylamine and hydrazine but as inhibitors of the hydrolytic process and not as acyl acceptors. Altogether, the results point out the crucial role of the carboxyl group of Glu268 in preventing nucleophiles, other than water, from binding as efficient acyl acceptors. This may also explain why CoA-dependent ALDHs never possesses a glutamate residue at position 268.     

Role of glutamate-268 in the catalytic mechanism of nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase from Streptococcus mutans

Nonphosphorylating nicotinamide adenine dinucleotide (phosphate)- [NAD(P)-] dependent aldehyde dehydrogenases share a number of conserved amino acid residues, several of which are directly implicated in catalysis. In the present study, the role of Glu-268 from nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN) from Streptococcus mutans was investigated. Its substitution by Ala resulted in a k(cat) decrease by 3 orders of magnitude. Pre-steady-state analysis showed that, for both the wild-type and E268A GAPNs, the rate-limiting step of the reaction is associated with deacylation. The pH dependence of the rate of acylation of wild-type GAPN is characterized by the contributions of distinct enzyme protonic species with two pK(a)s of 6.2 and 7.5. Substitution of Glu-268 by Ala resulted in a monosigmoidal pH dependence of the rate constant of acylation with a pK(a) of 6.2, which suggested the assignment of pK(a) 7.5 to Glu-268. Moreover, the E268A substitution did not significantly affect the efficiency of acylation of GAPN, showing that Glu-268 is not critically involved in the acylation, which includes Cys-302 nucleophilic activation and hydride transfer. On the contrary, the drastic decrease of the steady-state rate constant for the E268A GAPN demonstrated the essential role of Glu-268 in the deacylation. At basic pH, the solvent isotope effect of 2.3, characterized by a unique pK(a) of 7.7, and the linearity of the proton inventory showed that the rate-limiting process for deacylation is associated with the hydrolysis step and suggested that the glutamate form of Glu-268 acts as a base catalyst in this process. Surprisingly, the double-sigmoidal form of the pH-steady-state rate constant profile, characterized by pK(a) values of 6.1 and 7.4, revealed the high efficiency of the deacylation even at pH lower than 7.4. Therefore, we propose that the major role of Glu-268 is to promote deacylation through activation and orientation of the attacking water molecule, and in addition to act as a base catalyst at basic pH. From these results in relation to those recently described [Marchal, S., and Branlant, G. (1999) Biochemistry 38, 12950-12958], a scenario for the chemical catalysis of GAPN is proposed.     

Alkylation of acetylcholinesterase anionic centre with aziridinium ion accelerates the enzyme acylation step

Affinity labelling of acetylcholinesterase (EC 3.1.1.7, acetylcholine acetylhydrolase) anionic centre with N,N-dimethyl-2-phenylaziridinium ion accelerates the hydrolysis of non-ionic acetic esters by increasing the rate of the enzyme acylation step at least 500-times while the rate of the deacylation step remains unchanged. Simultaneously, at least a 10-fold decrease of the substrate binding affinity takes place. The acceleration phenomenon can be explained by the "induced fit" mechanism as the binding of the cationic label to the enzyme anionic site brings the esteratic centre into conformation which provides extra stabilization for the transition state of the enzyme acylation reaction, probably by a more close structural fit between the substrate molecule and the enzyme active centre.     

Fatty acid remodelling of phosphatidylinositol under conditions of de novo synthesis in rat liver microsomes

Phosphatidylinositol (PI) is initially synthesized in mammalian cells with a fatty acid composition similar to that of its precursor, primarily monounsaturated forms of cytidine diphosphodiglyceride (CDP-DAG). However, at the steady state, over 80% of PI exists in the 1-stearoyl, 2-arachidonoyl form. The fatty acid remodelling of PI is due to a number of deacylation/reacylation mechanisms. In the preceding paper we demonstrated that de novo synthesized PI is rapidly deacylated and subsequently reacylated. In this report we present further evidence that cycles of deacylation and reacylation are involved in the remodelling of PI. Incubation of microsomes with CDP-DAG of different fatty acid composition results in quantitative and qualitative differences in lysoPI formation. Additionally, analyses of the resulting lysoPI and PI species reveal that multiple species of fatty acids are incorporated into the 1-position of both PI and lysoPI. Addition of acylation cofactors (fatty acyl CoAs or ATP plus CoA) potentiate reacylation in this system. The addition of stearoyl or myristoyl CoA during de novo synthesis of PI results in the incorporation of these added fatty acids into the I-positive of PI. In addition, some evidence is presented that multiple mechanisms for remodelling of the 1-position of PI may be active in the microsomes, including ATP- and CoA-dependent acylation, ATP-independent, CoA-dependent acylation and CoA-independent mechanisms. Finally, the disappearance of only a subset of lysoPI species upon the addition of acylation cofactors suggests that the reacylation step exhibits some substrate specificity.