Biomedical subjects

C Kleanthous

Publications and source records attributed to C Kleanthous.

63 records · Page 4Linked to original sources

PubMed1992

Mechanistic and active-site studies on D(--)-mandelate dehydrogenase from Rhodotorula graminis.

D(--)-Mandelate dehydrogenase, the first enzyme of the mandelate pathway in the yeast Rhodotorula graminis, catalyses the NAD(+)-dependent oxidation of D(--)-mandelate to phenylglyoxylate. D(--)-2-(Bromoethanoyloxy)-2-phenylethanoic acid ['D(--)-bromoacetylmandelic acid'], an analogue of the natural substrate, was synthesized as a probe for reactive and accessible nucleophilic groups within the active site of the enzyme. D(--)-Mandelate dehydrogenase was inactivated by D(--)-bromoacetylmandelate in a psuedo-first-order process. D(--)-Mandelate protected against inactivation, suggesting that the residue that reacts with the inhibitor is located at or near the active site. Complete inactivation of the enzyme resulted in the incorporation of approx. 1 mol of label/mol of enzyme subunit. D(--)-Mandelate dehydrogenase that had been inactivated with 14C-labelled D(--)-bromoacetylmandelate was digested with trypsin; there was substantial incorporation of 14C into two tryptic-digest peptides, and this was lowered in the presence of substrate. One of the tryptic peptides had the sequence Val-Xaa-Leu-Glu-Ile-Gly-Lys, with the residue at the second position being the site of radiolabel incorporation. The complete sequence of the second peptide was not determined, but it was probably an N-terminally extended version of the first peptide. High-voltage electrophoresis of the products of hydrolysis of modified protein showed that the major peak of radioactivity co-migrated with N tau-carboxymethylhistidine, indicating that a histidine residue at the active site of the enzyme is the most likely nucleophile with which D(--)-bromoacetylmandelate reacts. D(--)-Mandelate dehydrogenase was incubated with phenylglyoxylate and either (4S)-[4-3H]NADH or (4R)-[4-3H]NADH and then the resulting D(--)-mandelate and NAD+ were isolated. The enzyme transferred the pro-R-hydrogen atom from NADH during the reduction of phenylglyoxylate. The results are discussed with particular reference to the possibility that this enzyme evolved by the recruitment of a 2-hydroxy acid dehydrogenase from another metabolic pathway.

Affinity Labels↗

PubMed1991

Stabilization of the shikimate pathway enzyme dehydroquinase by covalently bound ligand.

Reversible binding of a ligand to an enzyme active site can elicit a variety of changes in the protein, such as conformational changes (close to the site of binding or communicated over long distances), changes in the ionization state of surrounding amino acid side chains, changes in the interaction of the target protein with other subunits (or other proteins), or even changes in the thermodynamic stability of the protein. Relatively little attention has been given to studying these effects in proteins to which the ligand has been irreversibly bound, yet this can be a convenient way of studying the effects of ligand binding in the absence of association/dissociation equilibria. We report the dramatic changes which occur to the shikimate pathway enzyme dehydroquinase when ligand is attached to its active site after borohydride reduction of the mechanistically important Schiff's base intermediates. The effects of this modification have been characterized by limited proteolysis, circular dichroism, guanidine hydrochloride denaturation, and differential scanning calorimetry. The conclusions from these studies are that although anchoring the ligand at the active site does not cause a gross change in conformation, it does increase markedly the conformational stability of the protein. This is conclusively established by three separate experiments: 1) the modified protein is completely resistant to proteases, whereas the unmodified protein is very susceptible to proteolysis; 2) the concentration of guanidine hydrochloride required to unfold the ligand-linked dehydroquinase is 3-4-fold greater than that of the unmodified protein; 3) the melting temperature (Tm) of the modified protein is 40 degrees C higher than that of the unmodified protein. These results are a very clear example of the thermodynamic link between ligand binding, conformational stability, and proteolytic susceptibility in vitro and will be a useful system for dissecting the contributions of individual protein-ligand interactions to these parameters.

Calorimetry, Differential Scanning↗

PubMed1990

Active site labeling of the shikimate pathway enzyme, dehydroquinase. Evidence for a common substrate binding site within dehydroquinase and dehydroquinate synthase.

Dehydroquinase, the third enzyme of the shikimate biosynthetic pathway, is inactivated by iodoacetate. Iodoacetate behaves as an affinity label for the Escherichia coli enzyme with a Ki of 30 mM and a limiting inactivation rate of 0.014 min-1 at pH 7.0 and 25 degrees C. Affinity labeling is mediated by the negative charge of the reagent since iodoacetamide does not inactivate the enzyme. 2.1-2.3 mol of carboxymethyl groups are incorporated per mol of protein monomer resulting in 90% inactivation of enzymic activity. The majority of the bound label (80%) is split equally between 2 methionine residues, Met-23 and Met-205, which were identified by sequencing radiolabelled peptide fragments isolated after proteolytic digestion. An equilibrium mixture of the substrate (dehydroquinate) and product (dehydroshikimate) substantially reduces the inactivation rate and specifically decreases the incorporation of label at both of these site, implicating them as being in or near the active site of the enzyme. Sequence alignments with other biosynthetic dehydroquinases show that of the 2 methionine residues only Met-205 is conserved. N-terminal alignments of all the available dehydroquinase sequences (both catabolic and biosynthetic classes) revealed that Met-23, although itself not conserved, resides within a cluster of conserved sequence which may constitute part of the dehydroquinate binding site. A consensus sequence was derived from these alignments and used to probe the protein sequence data banks. A related sequence was found in dehydroquinate synthase, the enzyme which precedes dehydroquinase in the shikimate pathway. These results suggest that we have identified part of the dehydroquinate binding site in both enzymes.

Affinity Labels↗

PubMed1990

Reversible alkylation of an active site methionine residue in dehydroquinase.

Iodoacetic acid inactivates dehydroquinase by simultaneously alkylating 2 methionine residues (Met-23 and Met-205), presumed to be active site residues (described in Kleanthous, C., Campbell, D. G., and Coggins, J. R. (1990) J. Biol. Chem. 265, 10929-10934). Although both sites are carboxymethylated to the same degree in the inactivated enzyme, the modification of Met-205 may be reversed by treatment with mercaptoethanol at alkaline pH, as shown by the stoichiometric loss of label from this site. This, in turn, leads to partial reactivation of the inactive enzyme. Alkylation of Met-23 is not reversible under these conditions. The chemistry of the cleavage reaction at Met-205 was investigated by isolating the cleavage product which was identified by mass spectrometry as the ammonium salt of 2-hydroxyethyl thioacetate. This result is consistent with nucleophilic attack by the thiolate anion of mercaptoethanol on the alpha-carbon of the carboxymethyl moiety, which restores the side chain of the methionine residue (Met-205) and liberates 2-hydroxyethyl thioacetate. The differential reactivity of the 2 carboxymethylated methionine residues toward mercaptoethanol is likely to be a reflection of their different microenvironments in the folded protein. This assertion is borne out by unfolding experiments which indicate that neither of the carboxymethylated methionine residues in dicarboxymethylated dehydroquinase is susceptible to mercaptoethanol cleavage if the protein is first denatured by either guanidine hydrochloride or urea. Furthermore, this denatured material refolds after removal of denaturant to yield protein with reactivation properties similar to untreated, dicarboxymethylated enzyme.

Affinity Labels↗

PubMed1989

Identification of a cysteine residue at the active site of Escherichia coli isocitrate lyase.

Escherichia coli isocitrate lyase was inactivated by iodacetate in a pseudo-first-order process. Complete inactivation was associated with the incorporation of only one carboxymethyl group per enzyme subunit. The substrate and products of the enzyme protected against inactivation, suggesting that the reactive group may be located at the active site. Isolation and sequencing of a carboxymethylated peptide showed that the modified residue was a cysteine, in the sequence Cys-Gly-His-Met-Gly-Gly-Lys. The reactivity of isocitrate lyase to iodoacetate declined with pH, following a titration curve for a group of pKa 7.1. The Km of the enzyme for isocritrate declined over the same pH range.

Amino Acid Sequence↗

PubMed1988

Substitutions in the active site of chloramphenicol acetyltransferase: role of a conserved aspartate.

The role of conserved Asp-199 in chloramphenicol acetyltransferase (CAT) has been investigated by site-directed mutagenesis. Substitution of Asp-199 by alanine results in a thermolabile mutant enzyme (Ala-199 CAT) with reduced kcat(13-fold) but similar Km values to wild type CAT. Replacement by asparagine gives rise to a thermostable mutant enzyme (Asn-199 CAT) with much reduced kcat(1500-fold). Furthermore, Asn-199 CAT shows anomalous inactivation kinetics with the affinity reagent 3-(bromo-acetyl)chloramphenicol. These results favor a structural role for Asp-199 rather than a catalytic one, in keeping with crystallographic evidence for involvement of Asp-199 in a tight salt bridge with Arg-18. Replacement of Arg-18 by valine results in a mutant enzyme (Val-18 CAT) with similar properties to Ala-199 CAT. The catalytic imidazole of His-19 appears to be conformationally constrained by hydrogen bonding between N1-H and the carbonyl oxygen of the same residue and by ring stacking with Tyr-25.

Alanine↗

PubMed1988

The role of an active site histidine in the catalytic mechanism of aspartate transcarbamoylase.

Although the allosteric enzyme aspartate transcarbamoylase from Escherichia coli has been the focus of numerous physical and enzymological studies for over 2 decades, the catalytic mechanism is still poorly understood. There has been much speculation regarding the role of a conserved histidine residue at position 134 which recent crystallographic studies have implicated in the catalytic mechanism as a general acid or as a general base. We have used a combination of site-directed mutagenesis, 13C-isotope incorporation, and high field NMR to probe the role of His134 in the catalytic mechanism of aspartate transcarbamoylase. By comparing the wild-type catalytic trimer with that from a partially active mutant in which His134 is replaced by alanine, we have assigned the 13C resonance for His134 in the wild-type enzyme. This residue is shown to have a pK less than 6, indicating that the imidazole ring is unprotonated at pH values optimal for enzymatic activity (pH 8.0). This result eliminates the possibility of His134 participating as a general acid in the carbamoyl transfer mechanism. Since the crystallographic studies indicate that His134 is close enough to hydrogen-bond to the carbonyl of the liganded bisubstrate analog N-(phosphonacetyl)-L-aspartate, the imidazole ring would be oriented as to make it unlikely that the N1 lone pair of electrons could participate in general base catalysis. Moreover, if His134 is implicated in base catalysis, we would have expected a much greater loss of activity upon its replacement by alanine. Perhaps the role of His134 is merely to help position the carbonyl group of carbamoyl phosphate for nucleophilic attack by the alpha-amino group of aspartate.

Aspartate Carbamoyltransferase↗

PubMed1985

3-(Bromoacetyl)chloramphenicol, an active site directed inhibitor for chloramphenicol acetyltransferase.

Bacterial resistance to the antibiotic chloramphenicol is normally mediated by chloramphenicol acetyltransferase (CAT), which utilizes acetyl coenzyme A as the acyl donor in the inactivation reaction. 3-(Bromoacetyl)chloramphenicol, an analogue of the acetylated product of the forward reaction catalyzed by CAT, was synthesized as a probe for accessible and reactive nucleophilic groups within the active site. Extremely potent covalent inhibition was observed. Affinity labeling was demonstrated by the protection afforded by chloramphenicol at concentrations approaching Km for the substrate. Inactivation was stoichiometric, 1 mol of the inhibitor covalently bound per mole of enzyme monomer, with complete loss of both the acetylation and hydrolytic activities associated with CAT. N3-(Carboxymethyl)histidine was identified as the only alkylated amino acid, implicating the presence of a unique tautomeric form of a reactive imidazole group at the catalytic center. The proteolytic digestion of CAT modified with 3-(bromo[14C]-acetyl)chloramphenicol yielded three labeled peptide fractions separable by reverse-phase high-pressure liquid chromatography. Each peptide fraction was sequenced by fast atom bombardment mass spectrometry; the labeled peptide in each case was found to span the highly conserved region in the primary structure of CAT, which had been tentatively assigned as the active site. The rapid, stoichiometric, and specific alkylation of His-189, taken together with the high degree of conservation of the adjacent amino acid residues, strongly suggests a central role for His-189 in the catalytic mechanism of CAT.

Acetyltransferases↗

PubMed1984

Analysis of the mechanism of chloramphenicol acetyltransferase by steady-state kinetics. Evidence for a ternary-complex mechanism.

The mechanism of the enzymic reaction responsible for chloramphenicol resistance in bacteria was examined by steady-state kinetic methods. The forward reaction catalysed by chloramphenicol acetyltransferase leads to inactivation of the antibiotic. Use of alternative acyl donors and acceptors, as well as the natural substrates, has yielded data that favour the view that the reaction proceeds to the formation of a ternary complex by a rapid-equilibrium mechanism wherein the addition of substrates may be random but a preference for acetyl-CoA as the leading substrate can be detected. Chloramphenicol and acetyl-CoA bind independently, but the correlation between directly determined and kinetically derived dissociation constants is imperfect because of an unreliable slope term in the rate equation. The reverse reaction, yielding acetyl-CoA and chloramphenicol, was studied in a coupled assay involving citrate synthase and malate dehydrogenase, and is best described by a rapid-equilibrium mechanism with random addition of substrates. The directly determined dissociation constant for CoA is in agreement with that derived from kinetic measurements under the assumption of an independent-sites model.

Acetyl Coenzyme A↗