Effect of serine hydroxamate on the growth of Escherichia coli

The structural analogue of l-serine, l-serine hydroxamate, inhibited the growth of Escherichia coli K-12. Of the other amino acid hydroxamates tested, only l-lysine hydroxamate reduced the rate of growth. Inhibition of growth by l-serine hydroxamate was rapidly reversed by the addition of l-serine to the bacterial culture or by removal of the analogue by filtration. The reversal of inhibition was specific for l-serine. l-Alanine, glycine, or adenine had no effect on an inhibited culture. No evidence for active transport of the analogue was obtained.     

Roles of l-serine and sphingolipid synthesis in brain development and neuronal survival

Sphingolipids represent a class of membrane lipids that contain a hydrophobic ceramide chain as its common backbone structure. Sphingolipid synthesis requires two simple components: l-serine and palmitoyl CoA. Although l-serine is classified as a non-essential amino acid, an external supply of l-serine is essential for the synthesis of sphingolipids and phosphatidylserine (PS) in particular types of central nervous system (CNS) neurons. l-Serine is also essential for these neurons to undergo neuritogenesis and to survive. Biochemical analysis has shown that l-serine is synthesized from glucose and released by astrocytes but not by neurons, which is the major reason why this amino acid is an essential amino acid for neurons. Biosynthesis of membrane lipids, such as sphingolipids, PS, and phosphatidylethanolamine (PE), in neurons is completely dependent on this astrocytic factor. Recent advances in lipid biology research using transgenic mice have demonstrated that synthesis of endogenous l-serine and neuronal sphingolipids is essential for brain development. In this review, we discuss the metabolic system that coordinates sphingolipid synthesis with the l-serine synthetic pathway between neurons and glia. We also discuss the crucial roles of the metabolic conversion of l-serine to sphingolipids in neuronal development and survival. Human diseases associated with serine and sphingolipid biosynthesis are also discussed.     

Effects of acetate and other short-chain fatty acids on sugar and amino acid uptake of Bacillus subtilis

Acetate and other short chain n-fatty acids (C(1)-C(6)) inhibit strongly the uptake of l-serine or other l-amino acids but inhibit only weakly that of alpha-methylglucoside or fructose, whether measured in whole cells of Bacillus subtilis or in membrane vesicles that have been energized with reduced nicotinamide adenine dinucleotide (NADH), l-alpha-glycerol phosphate, or ascorbate plus phenazine methosulfate. The acetate inhibition is noncompetitive, as was shown for l-alpha-aminoisobutyric acid uptake by whole cells and for l-serine uptake by membrane vesicles. In membrane preparations, neither NADH oxidation nor the reduction of cytochromes by NADH are affected by fatty acids. All of these effects are similar to those of 2, 4-dinitrophenol. It is concluded that the fatty acids "uncouple" the amino acid carrier proteins from the cytochrome-linked electron transport system (to which they may be coupled via protein interaction or via a cation gradient).     

Kinetic Evidence of a Noncatalytic Substrate Binding Site That Regulates Activity in Legionella pneumophilal-Serine Dehydratase

The l-serine dehydratase from Legionella pneumophila (lpLSD) has recently been shown to contain a domain (β domain) that has a high degree of structural homology with the ASB domain of d-3-phosphoglycerate dehydrogenase (PGDH) from Mycobacterium tuberculosis. Furthermore, this domain has been shown by sequence homology to be present in all bacterial l-serine dehydratases that utilize an Fe-S catalytic center. In PGDH, l-serine binds to the ACT domain to inhibit catalytic activity. However, substrate must be bound to the ASB domain for serine to exert its effect. As such, the ASB domain acts as a codomain for the action of l-serine. Pre-steady-state kinetic analysis of l-serine binding to lpLSD demonstrates that l-serine binds to a second noncatalytic site and produces a conformational change in the enzyme. The rate of this conformational change is too slow for its participation in the catalytic cycle but rather occurs prior to catalysis to produce an activated form of the enzyme. That the conformational change must occur prior to catalysis is shown by a lag in the production of product that exhibits essentially the same rate constant as the conformational change. The second, noncatalytic site for l-serine is likely to be the ASB domain (β domain) of lpLSD that functions in a manner similar to that in PGDH. A mechanism whose overall effect is to keep l-serine levels from accumulating to high levels while not completely depleting the l-serine pool in the bacterial cell is proposed.     

L-serine deaminase of Escherichia coli

The native l-serine deaminase (l-serine hydrolyase, deaminating, EC 4.2.1.13) of Escherichia coli K-12, which seems to be a very labile protein, is rather stable in concentrated solution. Dilution rapidly inactivates it, but in the presence of a saturating concentration of l-serine the molecule is protected from inactivation. It is a very specific enzyme; l-serine is the sole substrate with a K(m) value of 6.60 x 10(-3)m. d-Serine and l-cysteine are competitive inhibitors. Substrate saturation curves of the native enzyme show sigmoid shape, whereas the enzyme liberated from the bacteria in the presence of l-serine exhibits normal Michaelis-Menten kinetics.     

Pimelic diphenylamide 106 is a slow, tight-binding inhibitor of class I histone deacetylases

Histone deacetylase (HDAC) inhibitors, including various benzamides and hydroxamates, are currently in clinical development for a broad range of human diseases, including cancer and neurodegenerative diseases. We recently reported the identification of a family of benzamide-type HDAC inhibitors that are relatively non-toxic compared with the hydroxamates. Members of this class of compounds have shown efficacy in cell-based and mouse models for the neurodegenerative diseases Friedreich ataxia and Huntington disease. Considerable differences in IC(50) values for the various HDAC enzymes have been reported for many of the HDAC inhibitors, leading to confusion as to the HDAC isotype specificities of these compounds. Here we show that a benzamide HDAC inhibitor, a pimelic diphenylamide (106), is a class I HDAC inhibitor, demonstrating no activity against class II HDACs. 106 is a slow, tight-binding inhibitor of HDACs 1, 2, and 3, although inhibition for these enzymes occurs through different mechanisms. Inhibitor 106 also has preference toward HDAC3 with K(i) of approximately 14 nm, 15 times lower than the K(i) for HDAC1. In comparison, the hydroxamate suberoylanilide hydroxamic acid does not discriminate between these enzymes and exhibits a fast-on/fast-off inhibitory mechanism. These observations may explain a paradox involving the relative activities of pimelic diphenylamides versus hydroxamates as gene activators.     

Kinetic, mutagenic, and structural homology analysis of L-serine dehydratase from Legionella pneumophila

A structural database search has revealed that the same fold found in the allosteric substrate binding (ASB) domain of Mycobacterium tuberculosis D-3-phosphoglycerate dehydrogenase (PGDH) is found in l-serine dehydratase from Legionella pneumophila. The M. tuberculosis PGDH ASB domain functions in the control of catalytic activity. Bacterial l-serine dehydratases are 4Fe-4S proteins that convert l-serine to pyruvate and ammonia. Sequence homology reveals two types depending on whether their α and β domains are on the same (Type 2) or separate (Type 1) polypeptides. The α domains contain the catalytic iron-sulfur center while the β domains do not yet have a described function, but the structural homology with PGDH suggests a regulatory role. Type 1 β domains also contain additional sequence homologous to PGDH ACT domains. A continuous assay for l-serine dehydratase is used to demonstrate homotropic cooperativity, a broad pH range, and essential irreversibility. Product inhibition analysis reveals a Uni-Bi ordered mechanism with ammonia dissociating before pyruvate. l-Threonine is a poor substrate and l-cysteine and d-serine are competitive inhibitors with K(i) values that differ by almost 10-fold from those reported for Escherichia colil-serine dehydratase. Mutagenesis identifies the three cysteine residues at the active site that anchor the iron-sulfur complex.     

Structural basis for acyl acceptor specificity in the achromobactin biosynthetic enzyme AcsD

Siderophores are known virulence factors, and their biosynthesis is a target for new antibacterial agents. A non-ribosomal peptide synthetase-independent siderophore biosynthetic pathway in Dickeya dadantii is responsible for production of the siderophore achromobactin. The D. dadantii achromobactin biosynthesis protein D (AcsD) enzyme has been shown to enantioselectively esterify citric acid with l-serine in the first committed step of achromobactin biosynthesis. The reaction occurs in two steps: stereospecific activation of citric acid by adenylation, followed by attack of the enzyme-bound citryl adenylate by l-serine to produce the homochiral ester. We now report a detailed characterization of the substrate profile and mechanism of the second (acyl transfer) step of AcsD enzyme. We demonstrate that the enzyme catalyzes formation of not only esters but also amides from the citryl-adenylate intermediate. We have rationalized the substrate utilization profile for the acylation reaction by determining the first X-ray crystal structure of a product complex for this enzyme class. We have identified the residues that are important for both recognition of l-serine and catalysis of ester formation. Our hypotheses were tested by biochemical analysis of various mutants, one of which shows a reversal of specificity from the wild type with respect to non-natural substrates. This change can be rationalized on the basis of our structural data. That this change in specificity is accompanied by no loss in activity suggests that AcsD and other members of the non-ribosomal peptide synthetase-independent siderophore superfamily may have biotransformation potential.     

Reversed hydroxamate-bearing thermolysin inhibitors mimic a high-energy intermediate along the enzyme-catalyzed proteolytic reaction pathway

A series of inhibitors that bear a reversed hydroxamate moiety have been evaluated as transition state analogue inhibitors for thermolysin. A linear correlation is observed between the K(i) values of these inhibitors and the kinetic parameters (K(M)/k(cat)) of the parallel series of related substrates, satisfying the criterion stipulated for transition state analogue inhibitors by Bartlett and Marlowe. Furthermore, examination of the binding mode of a related reversed hydroxamate bearing thermolysin inhibitor, in comparison with a transition state postulated for the enzyme-catalyzed proteolytic reaction revealed that the inhibitors under study mimic the electronic as well as the geometric characteristics of the transition state. On the basis of these results it may be concluded that the hydroxamate-bearing zinc protease inhibitors are a new type of transition state analogue inhibitors.     

Ligand-induced formation of a transient tryptophan synthase complex with αββ subunit stoichiometry

The prototypical tryptophan synthases form a stable heterotetrameric αββα complex in which the constituting TrpA and TrpB1 subunits activate each other in a bidirectional manner. The hyperthermophilic archaeon Sulfolobus solfataricus does not contain a TrpB1 protein but instead two members of the phylogenetically distinct family of TrpB2 proteins, which are encoded within (sTrpB2i) and outside (sTrpB2a) the tryptophan operon. It has previously been shown that sTrpB2a does not functionally or structurally interact with sTrpA, whereas sTrpB2i substantially activates sTrpA in a unidirectional manner. However, in the absence of catalysis, no physical complex between sTrpB2i and sTrpA could be detected. In order to elucidate the structural requirements for complex formation, we have analyzed the interaction between sTrpA (α-monomer) and sTrpB2i (ββ-dimer) by means of spectroscopy, analytical gel filtration, and analytical ultracentrifugation, as well as isothermal titration calorimetry. In the presence of the TrpA ligand glycerol 3-phosphate (GP) and the TrpB substrate l-serine, sTrpA and sTrpB2i formed a physical complex with a thermodynamic dissociation constant of about 1 μM, indicating that the affinity between the α- and ββ-subunits is weaker by at least 1 order of magnitude than the affinity between the corresponding subunits of prototypical tryptophan synthases. The observed stoichiometry of the complex was 1 subunit of sTrpA per 2 subunits of sTrpB2i, which corresponds to a αββ quaternary structure and testifies to a strong negative cooperativity for the binding of the α-monomers to the ββ-dimer. The analysis of the interaction between sTrpB2i and sTrpA in the presence of several substrate, transition state, and product analogues suggests that the αββ complex remains stable during the whole catalytic cycle and disintegrates into α- and ββ-subunits upon the release of the reaction product tryptophan. The formation of a transient tryptophan synthase complex, together with the observed low affinity of sTrpB2i for l-serine, couples the rate of tryptophan biosynthesis in S. solfataricus to the cytosolic availability of l-serine.     

Hydroxamate-induced spectral perturbations of cobalt Aeromonas aminopeptidase

The absorption spectrum of cobalt(II)-substituted Aeromonas aminopeptidase is markedly perturbed by the presence of equimolar concentrations of D-amino acid hydroxamates and acyl hydroxamates that have previously been shown to be powerful inhibitors of this enzyme (Wilkes, S. H., and Prescott, J. M. (1983) J. Biol. Chem. 258, 13517-13521). D-Valine hydroxamate produces the most distinctive perturbation, splitting the characteristic 527 nm absorption peak of the cobalt enzyme to form peaks at 564, 520, and 487 nm with molar extinction values of 126, 98, and 67 M-1 cm-1, respectively. A qualitatively similar perturbation, albeit with lower extinction values, results from the addition of D-leucine hydroxamate, whereas D-alanine hydroxamate perturbs the spectrum, but does not evoke the peak at 564 nm. In contrast, hydroxamates of L-valine and L-leucine in concentrations equi-molar to that of the enzyme produce only faint indications of change in the spectrum, but the hydroxamates of several other L-amino acids perturb the spectrum essentially independently of the identity of the side chain and in a qualitatively different manner from that of D-valine hydroxamate and D-leucine hydroxamate. At the high enzyme:substrate ratios used in the spectral experiments, L-leucine hydroxamate and L-valine hydroxamate proved to be rapidly hydrolyzed, hence their inability to perturb the spectrum of the cobalt-substituted enzyme during the time course of a spectral experiment. Values of kcat for L-amino acid hydroxamates, all of which are good reversible inhibitors of the hydrolysis of L-leucine-p-nitroanilide by Aeromonas aminopeptidase, were found to range from 0.01 min-1 to 5.6 min-1 for the native enzyme and from 0.27 min-1 to 108 min-1 for the cobalt-substituted enzyme; their km values toward the cobalt aminopeptidase range from 1.2 X 10(-7) M to 1.9 X 10(-5) M. The mutual exclusivity of binding for hydroxamate inhibitors and 1-butaneboronic acid, previously shown by kinetics (Baker, J. O., Wilkes, S. H., Bayliss, M. E., and Prescott, J. M. (1983) Biochemistry 22, 2098-2103), was reflected in the characteristic spectra produced by these two types of inhibitors.     

Chemical mechanism of the serine acetyltransferase from Haemophilus influenzae

The pH dependence of kinetic parameters was determined in both reaction directions to obtain information about the acid-base chemical mechanism of serine acetyltransferase from Haemophilus influenzae (HiSAT). The maximum rates in both reaction directions, as well as the V/K(serine) and V/K(OAS), decrease at low pH, exhibiting a pK of approximately 7 for a single enzyme residue that must be unprotonated for optimum activity. The pH-independent values of V(1)/E(t), V(1)/K(serine)E(t), V/K(AcCoA)E(t), V(2)/E(t), V(2)/K(OAS)E(t), and V/K(CoA)E(t) are 3300 +/- 180 s(-1), (9.6 +/- 0.4) x 10(5) M(-1) s(-1), 3.3 x 10(6) M(-1) s(-1), 420 +/- 50 s(-1), (2.1 +/- 0.5) x 10(4) M(-1) s(-1), and (4.2 +/- 0.7) x 10(5) M(-1) s(-1), respectively. The K(i) values for the competitive inhibitors glycine and l-cysteine are pH-independent. The solvent deuterium kinetic isotope effects on V and V/K in the direction of serine acetylation are 1.9 +/- 0.2 and 2.5 +/- 0.4, respectively, and the proton inventories are linear for both parameters. Data are consistent with a single proton in flight in the rate-limiting transition state. A general base catalytic mechanism is proposed for the serine acetyltransferase. Once acetyl-CoA and l-serine are bound, an enzymic general base accepts a proton from the l-serine side chain hydroxyl as it undergoes a nucleophilic attack on the carbonyl of acetyl-CoA. The same enzyme residue then functions as a general acid, donating a proton to the sulfur atom of CoASH as the tetrahedral intermediate collapses, generating the products OAS and CoASH. The rate-limiting step in the reaction at limiting l-serine levels is likely formation of the tetrahedral intermediate between serine and acetyl-CoA.     

N-hydroxy-2-(naphthalene-2-ylsulfanyl)-acetamide, a novel hydroxamic acid-based inhibitor of aminopeptidase N and its anti-angiogenic activity

In the course of our screening, N-hydroxy-2-(naphthalene-2-ylsulfanyl)-acetamide (1), which contains a metal-chelating hydroxamate group, has been identified as a potent inhibitor of aminopeptidase N (APN, EC 3.4.11.2). Compound 1 potently inhibited APN activity with a K(i) value of 3.5 microM. It also inhibited the basic fibroblast growth-factor-induced invasion of bovine aortic endothelial cells at low micromolar concentrations.     

Mechanism of inhibition of the beta-lactamase of Enterobacter cloacae P99 by 1:1 complexes of vanadate with hydroxamic acids

The class C beta-lactamase of Enterobacter cloacae P99 is competitively inhibited by low concentrations of 1:1 complexes of vanadate and hydroxamic acids. Structure-activity studies indicated that the hydroxamic acid functional group was essential to this inhibition. Both aryl and alkyl hydroxamic acids form inhibitory ternary complexes with vanadate and the enzyme, although, in certain cases of the latter, the inhibition may not be seen because of the low formation constants of the vanadate-hydroxamic acid complex. After all of the vanadate species present in solution had been taken into account, "real" K(i) values for the vanadate complexes could be determined. The K(i) value of the best of the inhibitors that were investigated, the 1:1 complex of vanadate with 4-nitrobenzohydroxamic acid, was 0.48 microM. Kinetics studies showed that the association and dissociation rate constants of this complex with the enzyme were 1.48 x 10(6) s(-1) M(-1) and 0.73 s(-1), respectively; the magnitude of the latter indicates covalent interaction of the complex with the enzyme. (51)V NMR and UV-vis spectra suggest that the structure of the vanadate complex bound to the enzyme may be very similar to that in solution. A (13)C NMR spectrum of the enzyme complex with 4-nitrobenzo[(13)C]hydroxamic acid and vanadate yields a coordination-induced shift (CIS) of 7.74 ppm. This is significantly larger than that of the vanadate complex in free solution (3.62 ppm), suggesting either, somewhat contrary to the (51)V and UV-vis spectra, greater interaction between vanadium and the hydroxamate carbonyl oxygen in the enzyme complex than in free solution or, more likely, polarization of the hydroxamate by interaction, e.g., hydrogen bonding, with the enzyme. Molecular modeling indicates that a pentacoordinated vanadate complex may well be able to snugly occupy the enzyme active site; Asn 152 is suitably placed to hydrogen bond to the hydroxamic acid oxygen atom. The experimental results are in accord with a model whereby the vanadate-hydroxamate-enzyme complex is a moderately good analogue of the transition state of the reaction of the beta-lactamase with phosphonate inhibitors.     

Nitroxide metabolites from alkylhydroxylamines and N-hydroxyurea derivatives resulting from reductive inhibition of soybean lipoxygenase.

One proposed mechanism of the inactivation of lipoxygenase by inhibitors is the reduction of the catalytically active ferric form of the enzyme to its ferrous form. Recent studies have shown that compounds containing the hydroxamate moiety are potent inhibitors of lipoxygenase. The hydroxamate portion of the inhibitor is thought to bind to iron at the catalytic site of the enzyme. We now report evidence that the NOH of the hydroxamate group of N-(4-chlorophenyl)-N-hydroxy-N'-(3-chlorophenyl)urea, N-[(E)-3-(3-phenoxyphenyl)prop-2-enyl]acetohydroxamic acid (BW A4C), and N-(1-benzo(b)thien-2-ylethyl)-N-hydroxyurea (Zileuton) is oxidized by lipoxygenase to form their corresponding nitroxides, which are directly detected by electron paramagnetic resonance spectroscopy. It is consistently found that the selected NOH-containing compounds, e.g. alkylhydroxylamines or N-hydroxyureas, are also oxidized by lipoxygenase to form their corresponding nitroxides.     

Peptidyl hydroxamic acids as methionine aminopeptidase inhibitors

A new class of methionine aminopeptidase (MetAP) inhibitors, which contain an internal hydroxamate (N-acyl-N-alkylhydroxylamine) core as the metal-chelating group, has been designed, synthesized, and tested. The compounds exhibited reversible, competitive inhibition against Escherichia coli MetAP as well as human MetAP-1 and MetAP-2. The most potent inhibitor had a K(i) value of 2.5 microM and >20-fold selectivity toward E. coli MAP.     

Effect of Serine Hydroxamate on Phospholipid Synthesis in Escherichia coli

Serine hydroxamate, which inhibits the charging of seryl-transfer ribonucleic acid, reduced the synthesis of phospholipid and nucleic acids in Escherichia coli. This effect was analogous to depriving amino acid auxotrophs of their nutritional requirement and appears to be a manifestation of the stringent response shown by rel(+) strains of E. coli. Amino acid starvation (serine or methionine) alone or serine hydroxamate treatment alone results in 60 to 80% inhibition of lipid accumulation, 90% inhibition of ribonucleic acid accumulation, and an increase in guanosine tetraphosphate (ppGpp). These three effects were reversed by addition of chloramphenicol (CM). A combination of serine starvation and serine hydroxamate treatment resulted in inhibition of lipid and RNA accumulation as well as an increase in ppGpp, but the consequences of the double block were not reversed by CM. We conclude that a strong interrelationship exists among these processes and that CM acts to relax a stringent response by mechanisms other than interference with ppGpp formation. All species of phospholipid were affected by a stringent response evoked by amino acid starvation or addition of serine hydroxamate, but in all cases the synthesis of phosphatidylethanolamine was most severely inhibited. Serine hydroxamate was not incorporated into lipid but specifically caused phosphatidylserine accumulation. Serine starvation produced a dramatic alteration of the distribution of isotope incorporated into phospholipid, which resulted from the stringent response compounded with the limitation of a substrate for phosphatidylserine synthesis.     

Carbonic anhydrase inhibitors: inhibition of cytosolic/tumor-associated isoforms I, II, and IX with iminodiacetic carboxylates/hydroxamates also incorporating benzenesulfonamide moieties

The synthesis of a new class of sulfonamide carbonic anhydrase (CA, EC 4.2.1.1) inhibitors (CAIs), also possessing carboxylate/hydroxamate moieties in their molecule, is reported. These compounds may act on dual antitumor targets, the tumor-associated CA isozymes (CA IX) and some matrix metalloproteinases (MMPs). The compounds were prepared by an original method starting from iminodiacetic acid, and assayed as inhibitors of three isozymes, hCA I, II (cytosolic), and IX (transmembrane). The new derivatives showed weak inhibitory activity against isozyme I (K(I)s in the range of 95-8300 nM), were excellent to moderate CA II inhibitors (K(I)s in the range of 8.4-65 nM), and very good and selective CA IX inhibitors (K(I)s in the range of 3.8-26 nM). The primary sulfonamide moiety is a better zinc-binding group in the design of CAIs as compared to the carboxylate/hydroxamate one, but the presence of hydroxamate functionalities in the molecule of CAIs leads to selectivity for the tumor-associated isozyme IX over the ubiquitous, cytosolic isoform II.     

Spectroscopic characterisation of n-octanohydroxamic acid and potassium hydrogen n-octanohydroxamate

The crystal structure and spectroscopic characteristics of n-octanohydroxamic acid and the potassium compound of that acid have been investigated by XRD, XPS, FTIR and Raman spectroscopy. XRD revealed that the acid is in the keto Z conformation with the alkyl chains oriented along the z-direction and hydrogen bonding between hydroxamate moieties. Vibrational spectra confirm this conclusion. Chemical analysis, XRD and XPS established that the potassium compound is the acid salt KH(C₇H₉CONO)₂. The crystal structure showed that the hydroxamate groups are also in the keto Z conformation and this is supported by vibrational spectra. In the acid salt, the two hydroxamate moieties are connected by a symmetrical O-H-O short hydrogen bonded linkage between the two hydroxamate oxygen atoms and this explains the absence of a discernible O-H stretch band in the vibrational spectra. Identification of the vibrational bands displayed is supported by deuteration and ¹⁵N substitution.     

Inhibitor-mediated stabilization of the conformational structure of a histone deacetylase-like amidohydrolase

Histone deacetylases are major regulators of eukaryotic gene expression. Not unexpectedly, histone deacetylases are among the most promising targets in cancer therapy. However, despite huge efforts in histone deacetylase inhibitor design, very little is known about the impact of histone deacetylase inhibitors on enzyme stability. In this study, the conformational stability of a well-established histone deacetylase homolog with high structural similarity (histone deacetylase-like amidohydrolase from Bordetella/Alcaligenes species FB188) was investigated using denaturation titrations and stopped-flow kinetics. Based on the results of these complementary approaches, we conclude that the interconversion of native histone deacetylase-like amidohydrolase into its denatured form involves several intermediates possessing different enzyme activities and conformational structures. The refolding kinetics has shown to be strongly dependent on Zn(2+) and to a lesser extent on K(+), which underlines their importance not only for catalytic function but also for maintaining the correct conformational structure of the enzyme. Two main unfolding processes of histone deacetylase-like amidohydrolase were differentiated. The unfolding occurring at submolar concentrations of the denaturant guanidine hydrochloride was not affected by inhibitor binding, whereas the unfolding at higher concentrations of guanidine hydrochloride was strongly affected. It was shown that the known inhibitors suberoylanilide hydroxamic acid and cyclopentylpropionyl hydroxamate are capable of stabilizing the conformational structure of histone deacetylase-like amidrohydrolase. Judging from the free energies of unfolding, the protein stability was increased by 9.4 and 5.4 kJ.mol(-1) upon binding of suberoylanilide hydroxamic acid and cyclopentylpropionyl hydroxamate, respectively.