Multi-step analysis of Hg2+ ion inhibition of jack bean urease

We performed a multi-step analysis of the inhibition of jack bean urease by Hg(2+) ions that included residual activity measurements after incubation of the enzyme with the metal ion, reactivation of Hg(2+)-inhibited urease, protection of urease with thiol reagents prior to incubation with Hg(2+), progress curve analysis, and spectroscopic assay of thiol groups in urease-Hg(2+) complexes with a cysteine selective agent 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). Hg(2+) ions were found to form stable complexes with urease that could rapidly be reversed only by the treatment with dithiotreitol, and not by dilution or dialysis. The residual activity data interpreted in terms of the Hill equation revealed the multisite Hg(2+) inhibition of urease, and along with the DTNB thiol-assay they demonstrated the involvement in the reaction with Hg(2+) of six cysteine residues per enzyme subunit, including the active-site flap cysteine. The molar ratios of the inhibitor and enzyme imply that the inhibition consists of the formation of RSHgX complexes, X being a water molecule or an anion. The time-dependent Hg(2+) inhibitory action on urease determined in the system without enzyme preincubation was best described by slow-binding mechanism with the steady-state inhibition constant K(i) = 1.9 nM (+/-10%).     

Reactivity of the essential thiol of Klebsiella aerogenes urease. Effect of pH and ligands on thiol modification

The kinetics of Klebsiella aerogenes urease inactivation by disulfide and alkylating agents was examined and found to follow pseudo-first-order kinetics. Reactivity of the essential thiol is affected by the presence of substrate and competitive inhibitors, consistent with a cysteine located proximal to the active site. In contrast to the results observed with other reagents, the rate of activity loss in the presence of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) saturated at high reagent concentrations, indicating that DTNB must first bind to urease before inactivation can occur. The pH dependence for the rate of urease inactivation by both disulfide and alkylating agents was consistent with an interaction between the thiol and a second ionizing group. The resulting macroscopic pKa values for the 2 residues are less than 5 and 12. Spectrophotometric studies at pH 7.75 demonstrated that 2,2'-dithiodipyridine (DTDP) modified 8.5 +/- 0.2 mol of thiol/mol of enzyme or 4.2 mol of thiol/mol of catalytic unit. With the slow tight binding competitive inhibitor phenyl-phosphorodiamidate (PPD) bound to urease, 1.1 +/- 0.1 mol of thiol/mol of catalytic unit were protected from modification. PPD-bound DTDP-modified urease could be reactivated by dialysis, consistent with the presence of one thiol per active site. Analogous studies at pH 6.1, using the competitive inhibitor phosphate, confirmed the presence of one protected thiol per catalytic unit. Under denaturing conditions, 25.5 +/- 0.3 mol of thiol/mol of enzyme (Mr = 211, 800) were modified by DTDP.     

Mono- (Ag, Hg) and di- (Cu, Hg) valent metal ions effects on the activity of jack bean urease. Probing the modes of metal binding to the enzyme

The inhibition of urease by heavy metal ions has been habitually ascribed to the reaction of the ions with enzyme thiol groups, resulting in the formation of mercaptides. To probe the modes of metal binding to the enzyme, in this work the reaction of mono- (Ag, Hg) and di- (Cu, Hg) valent metal ions with jack bean urease was studied. The enzyme was reacted with different concentrations of the metal ions for different periods of times, when its residual activity was assayed and thiol content titrated. The titration carried out with DTNB was done to examine the involvement of urease thiol groups in metal ion binding. The binding was further probed by reactivation of the metal ion-enzyme complexes with DTT, EDTA and dilution. The results are discussed in terms of the HSAB concept. In inhibiting urease the metal ions showed a common feature in that they inhibited the enzyme within a comparable micromolar range, and also in that their inhibition was multisite. By contrast, the main distinguishing feature in their action consisted of the involvement of enzyme thiol groups in the reaction. Hg (2+) and Hg2(2+) inhibition was found thoroughly governed by the reaction with the enzyme thiols, and the complete loss of enzyme activity involved all thiols available in the enzyme under non-denaturating conditions. In contrast, Ag+ and Cu2+ ions for the complete inactivation of the enzyme required 53 and 60% of thiols, respectively. Accordingly, Ag+ and Cu2+ binding to functional groups in urease other than thiols, i.e. N- and O-containing groups, cannot be excluded. Based on the reactivation experiments this seems particularly likely for Cu2+, whose concurrent binding to thiols and other groups might distort the architecture of the active site (the mechanism of which remains to be elucidated) resulting in the observed inhibitory effects.     

Structure of the Mature Streptococcal Cysteine Protease Exotoxin mSpeB in Its Active Dimeric Form

Invasive infections of Streptococcus pyogenes are dependent on the cysteine protease streptococcal pyrogenic exotoxin B. Previous structures of the enzyme have not disclosed the proper active-site configuration. Here, the crystal structure of the mature enzyme is presented to 1.55 Å, disclosing a homodimer. A serine from one subunit inserts into the active site of the other to donate to the oxyanion hole and coordinates the ligand proximal to the active-site cysteine. Dimerization is unique to the mature form and is clearly a prerequisite for catalysis. The present structure supports a tripartite switch system that is triggered upon dimerization and substrate binding: (1) liberation of the active-site histidine from an inactive configuration, (2) relocation of residues blocking the substrate binding pockets and (3) repositioning of two active-site tryptophans to settle in the active configuration. Based on the present structure, the active site of clan CA cysteine proteases is expanded and a detailed mechanism of the deacylation mechanism is proposed. The results may have applications for the development of protease inhibitors specific to bacterial cysteine proteases.     

A new proposal for urease mechanism based on the crystal structures of the native and inhibited enzyme from Bacillus pasteurii: why urea hydrolysis costs two nickels.

BACKGROUND: Urease catalyzes the hydrolysis of urea, the final step of organic nitrogen mineralization, using a bimetallic nickel centre. The role of the active site metal ions and amino acid residues has not been elucidated to date. Many pathologies are associated with the activity of ureolytic bacteria, and the efficiency of soil nitrogen fertilization with urea is severely decreased by urease activity. Therefore, the development of urease inhibitors would lead to a reduction of environmental pollution, to enhanced efficiency of nitrogen uptake by plants, and to improved therapeutic strategies for treatment of infections due to ureolytic bacteria. Structure-based design of urease inhibitors would require knowledge of the enzyme mechanism at the molecular level. RESULTS: The structures of native and inhibited urease from Bacillus pasteurii have been determined at a resolution of 2.0 A by synchrotron X-ray cryogenic crystallography. In the native enzyme, the coordination sphere of each of the two nickel ions is completed by a water molecule and a bridging hydroxide. A fourth water molecule completes a tetrahedral cluster of solvent molecules. The enzyme crystallized in the presence of phenylphosphorodiamidate contains the tetrahedral transition-state analogue diamidophosphoric acid, bound to the two nickel ions in an unprecedented mode. Comparison of the native and inhibited structures reveals two distinct conformations of the flap lining the active-site cavity. CONCLUSIONS: The mode of binding of the inhibitor, and a comparison between the native and inhibited urease structures, indicate a novel mechanism for enzymatic urea hydrolysis which reconciles the available structural and biochemical data.     

Structural and functional role of nickel ions in urease by molecular dynamics simulation

Nickel ions play several roles in the biological processes of microorganisms and plants. Urease has a nickel-containing active site and catalyzes the hydrolysis of urea to yield ammonia and carbamate. In the present study, the role of nickel ions is examined using molecular dynamics simulations of the holo and apo forms. Nonbonded models used for the nickel ions provide good reproduction of the active-site structure as indicated in the crystallized structure. The results confirm that urease has a rigid active site in either its holo or its apo form. A new conformation of the flap is observed in apo urease. The connection between the metal center and His^α323 is proposed to be responsible for maintaining the flap conformation. The binding free energy of acetohydroxamic acid and urease is estimated using the molecular mechanics–generalized Born/surface area method. The binding free energy is primarily driven by electrostatic interactions in the presence of nickel ions. Normal mode analysis is employed to characterize the movements of the flap in apo urease.     

Competitive inhibitors of Klebsiella aerogenes urease. Mechanisms of interaction with the nickel active site

We examined several compounds for their mechanisms of inhibition with the nickel-containing active site of homogeneous Klebsiella aerogenes urease. Thiolate anions competitively inhibit urease and directly interact with the metallocenter, as shown by the pH dependence of inhibition and by UV-visible absorbance spectroscopic studies. Cysteamine, which possesses a cationic beta-amino group, exhibited a high affinity for urease (Ki = 5 microM), whereas thiolates containing anionic carboxyl groups were uniformly poor inhibitors. Phosphate monoanion competitively inhibits a protonated form of urease with a pKa of less than 5. Both the thiolate and phosphate inhibition results are consistent with charge repulsion by an anionic group in the urease active site. Acetohydroxamic acid (AHA) was shown to be a slow-binding competitive inhibitor of urease. This compound forms an initial E.AHA complex which then undergoes a slow transformation to yield an E.AHA* complex; the overall dissociation constant of AHA is 2.6 microM. Phenylphosphorodiamidate, also shown to be a slow-binding competitive inhibitor, possesses an overall dissociation constant of 94 pM. The tight binding of phenylphosphorodiamidate was exploited to demonstrate the presence of two active sites per enzyme molecule. Urease contains 4 mol of nickel/mol enzyme, hence there are two nickel ions/catalytic unit. Each of the two slow-binding inhibitors are proposed to form complexes in which the inhibitor bridges the two active site nickel ions. The inhibition results obtained for K. aerogenes urease are compared with inhibition studies of other ureases and are interpreted in terms of a model for catalysis proposed for the jack bean enzyme (Dixon, N.E., Riddles, P.W., Gazzola, C., Blakely, R.L., and Zerner, B. (1980) Can. J. Biochem. 58, 1335-1344).     

Thiolation of low-Mr phosphotyrosine protein phosphatase by thiol-disulfides

Thiol-disulfides cause a time- and a concentration-dependent inactivation of the low-M(r) phosphotyrosine protein phosphatase (PTP). We demonstrated that six of eight enzyme cysteines have similar reactivity against 5,5'-dithiobis(nitrobenzoic acid): Their thiolation is accompanied by enzyme inactivation. The inactivation of the enzyme by glutathione disulfide also is accompanied by the thiolation of six cysteine residues. Inorganic phosphate, a competitive enzyme inhibitor, protects the enzyme from inactivation, indicating that the inactivation results from thiolation of the essential active-site cysteine of the enzyme. The inactivation is reversed by dithiothreitol. Although all PTPs have three-dimensional active-site structures very similar to each other and also have identical reaction mechanisms, the thiol group contained in the active site of low-M(r) PTP seems to have lower reactivity than that of other PTPs in the protein thiolation reaction.     

Synthesis of chloromethyl ketone derivatives of fatty acids. Their use as specific inhibitors of acetoacetyl-coenzyme A thiolase, cholesterol biosynthesis and fatty acid synthesis.

A general route for the synthesis of chloromethyl ketone derivatives of fatty acids is described. 5-Chloro-4-oxopentanoic acid, 7-chloro-6-oxoheptanoic acid, 9-chloro-8-oxononanoic acid and 11-chloro-10-oxoundecanoic acid were synthesized by this method and tested as covalent inhibitors of pig heart acetoacetyl-CoA thiolase. The K1 decreased by approx. 20-fold for each pair of methylenes added to the chain length, showing that the initial stage in inhibitor binding occurs at a non-polar region of the protein. This region is probably located at the enzyme active site, since inhibition was prevented by acetoacetyl-CoA or acetyl-CoA but not by CoA. The site of modification by chloromethyl ketone derivatives of fatty acids is restricted to a thiol group, since inactivation of the enzyme was prevented by reversible thiomethylation of the active-site thiol. In contrast, an amino-directed reagent, citraconic anhydride, still inactivated the enzyme, even when the active-site thiol was protected. Evidence that the enzyme thiol was particularly reactive came from studies on the pH-dependence of the alkylation reaction and thiol-competition experiments. Inhibition of the enzyme proceeded suprisingly well at acidic pH values and a 10(5) molar excess of external thiol over active-site thiol was required to prevent inhibition by 0.3 mM-9-chloro-8-oxononanoic acid. In addition to inhibiting isolated acetoacetyl-CoA thiolase, in hepatocytes the chloromethyl ketone derivatives of fatty acids also inhibited chloresterol synthesis, which uses this enzyme as an early step in the biosynthetic pathway. In isolated cells, the chloromethyl ketone derivatives of fatty acids were considerably less specific in their inhibitory action compared with 3-acetylenic derivatives of fatty acids, which act as suicide inhibitors of acetoacetyl-CoA thiolase. However, 9-chloro-8-oxononanoic acid was also an effective inhibitor of both hepatic cholesterol and fatty acid synthesis in mice in vivo, whereas the acetylenic fatty acid derivative, dec-3-ynoic acid, was completely ineffective. The effective inhibitory dose of 9-chloro-8-oxononanoic acid (2.5-5 mg/kg) was substantially lower than the estimated LD50 for the inhibitor (100 mg/kg).     

Ureolytic activity of Nitellopsis obtusa (Characeae)

The decomposition of urea by Nitellopsis obtusa from Characeae was investigated. The intact cells were exposed to the inhibition by two typical urease inhibitors: boric acid and fluoride ion, used as a criterion to define if urease or UAL-ase is responsible for the ureolytic activity of the algae. It was found that boric acid and fluoride ion are simple competitive and slow-binding competitive inhibitors of Nitellopsis obtusa enzyme respectively, which is the response characteristic of urease. The inhibition constants equal to 2.3 and 0.1 mM for boric acid and fluoride ion, when compared to those of jack bean urease, indicate that in the observed kinetic behaviour of Nitellopsis obtusa urease partake transport processes taking place in the intact cells.     

Thioredoxin A active-site mutants form mixed disulfide dimers that resemble enzyme-substrate reaction intermediates

Thioredoxin functions in nearly all organisms as the major thiol-disulfide oxidoreductase within the cytosol. Its prime purpose is to maintain cysteine-containing proteins in the reduced state by converting intramolecular disulfide bonds into dithiols in a disulfide exchange reaction. Thioredoxin has been reported to contribute to a wide variety of physiological functions by interacting with specific sets of substrates in different cell types. To investigate the function of the essential thioredoxin A (TrxA) in the low-GC Gram-positive bacterium Bacillus subtilis, we purified wild-type TrxA and three mutant TrxA proteins that lack either one or both of the two cysteine residues in the CxxC active site. The pure proteins were used for substrate-binding studies known as “mixed disulfide fishing” in which covalent disulfide-bonded reaction intermediates can be visualized. An unprecedented finding is that both active-site cysteine residues can form mixed disulfides with substrate proteins when the other active-site cysteine is absent, but only the N-terminal active-site cysteine forms stable interactions. A second novelty is that both single-cysteine mutant TrxA proteins form stable homodimers due to thiol oxidation of the remaining active-site cysteine residue. To investigate whether these dimers resemble mixed enzyme-substrate disulfides, the structure of the most abundant dimer, C32S, was characterized by X-ray crystallography. This yielded a high-resolution (1.5Å) X-ray crystallographic structure of a thioredoxin homodimer from a low-GC Gram-positive bacterium. The C32S TrxA dimer can be regarded as a mixed disulfide reaction intermediate of thioredoxin, which reveals the diversity of thioredoxin/substrate-binding modes.     

Crystal structures of cystathionine gamma-synthase inhibitor complexes rationalize the increased affinity of a novel inhibitor

Cystathionine gamma-synthase catalyzes the committed step of methionine biosynthesis. This pathway is unique to microorganisms and plants, rendering the enzyme an attractive target for the development of antimicrobials and herbicides. We solved the crystal structures of complexes of cystathionine gamma-synthase (CGS) from Nicotiana tabacum with inhibitors of different compound classes. The complex with the substrate analog dl-E-2-amino-5-phosphono-3-pentenoic acid verifies the carboxylate-binding function of Arg423 and identifies the phosphate-binding pocket of the active site. The structure shows the function of Lys165 in specificity determination and suggests a role for the flexible side-chain of Tyr163 in catalysis. The importance of hydrophobic interactions for binding to the active-site center is highlighted by the complex with 3-(phosphonomethyl)pyridine-2-carboxylic acid. The low affinity of this compound is due to the non-optimal arrangement of the functional groups binding to the phosphate and carboxylate-recognition site, respectively. The newly identified inhibitor 5-carboxymethylthio-3-(3'-chlorophenyl)-1,2,4-oxadiazol, in contrast, shows the highest affinity to CGS reported so far. This affinity is due to binding to an additional active-site pocket not used by the physiological substrates. The inhibitor binds to the carboxylate-recognition site, and its tightly bent conformation enables it to occupy the novel binding pocket between Arg423 and Ser388. The described structures suggest improvements for known inhibitors and give guidelines for the development of new lead compounds.     

Crystal structures of a ligand-free and malonate-bound human caspase-1: implications for the mechanism of substrate binding

Caspase-1, a mediator of the posttranslational processing of IL-1beta and IL-18, requires an aspartic acid in the P1 position of its substrates. The mechanisms of caspase-1 activation remain poorly understood despite numerous structures of the enzyme complexed with aspartate-based inhibitors. Here we report a crystal structure of ligand-free caspase-1 that displays dramatic rearrangements of loops defining the active site to generate a closed conformation that is incompatible with substrate binding. A structure of the enzyme complexed with malonate shows the protein in its open (active-site ligand-bound) conformation in which malonate reproduces the hydrogen bonding network observed in structures with covalent inhibitors. These results illustrate the essential function of the obligatory aspartate recognition element that opens the active site of caspase-1 to substrates and may be the determinant responsible for the conformational changes between ligand-free and -bound forms of the enzyme, and suggest a new approach for identifying novel aspartic acid mimetics.     

Structure of serine acetyltransferase in complexes with CoA and its cysteine feedback inhibitor

Serine acetyltransferase (SAT, EC 2.3.1.30) catalyzes the CoA-dependent acetylation of the side chain hydroxyl group of l-serine to form O-acetylserine, as the first step of a two-step biosynthetic pathway in bacteria and plants leading to the formation of l-cysteine. This reaction represents a key metabolic point of regulation for the cysteine biosynthetic pathway due to its feedback inhibition by cysteine. We have determined the X-ray crystal structure of Haemophilus influenzae SAT in complexes with CoA and its cysteine feedback inhibitor. The enzyme is a 175 kDa hexamer displaying the characteristic left-handed parallel beta-helix (LbetaH) structural domain of the hexapeptide acyltransferase superfamily of enzymes. Cysteine is bound in a crevice between adjacent LbetaH domains and underneath a loop excluded from the coiled LbetaH. The proximity of its thiol group to the thiol group of CoA derived from superimposed models of the cysteine and CoA complexes confirms that cysteine is bound at the active site. Analysis of the contacts of SAT with cysteine and CoA and the conformational differences that distinguish these complexes provides a structural basis for cysteine feedback inhibition, which invokes competition between cysteine and serine binding and a cysteine-induced conformational change of the C-terminal segment of the enzyme that excludes binding of the cofactor.     

Probes of the catalytic site of cysteine dioxygenase

The first major step of cysteine catabolism, the oxidation of cysteine to cysteine sulfinic acid, is catalyzed by cysteine dioxygenase (CDO). In the present work, we utilize recombinant rat liver CDO and cysteine derivatives to elucidate structural parameters involved in substrate recognition and x-ray absorption spectroscopy to probe the interaction of the active site iron center with cysteine. Kinetic studies using cysteine structural analogs show that most are inhibitors and that a terminal functional group bearing a negative charge (e.g. a carboxylate) is required for binding. The substrate-binding site has no stringent restrictions with respect to the size of the amino acid. Lack of the amino or carboxyl groups at the alpha-carbon does not prevent the molecules from interacting with the active site. In fact, cysteamine is shown to be a potent activator of the enzyme without being a substrate. CDO was also rendered inactive upon complexation with the metal-binding inhibitors azide and cyanide. Unlike many non-heme iron dioxygenases that employ alpha-keto acids as cofactors, CDO was shown to be the only dioxygenase known to be inhibited by alpha-ketoglutarate.     

Jack bean (Canavalia ensiformis) urease. Probing acid-base groups of the active site by pH variation.

A pH-variation study of jack bean (Canavalia ensiformis) urease steady-state kinetic parameters and of the inhibition constant of boric acid, a urease competitive inhibitor, was performed using both noninhibitory organic (MES, HEPES and CHES) and inhibitory inorganic (phosphate) buffers, in an effort to elucidate the functions exercised in the catalysis by the ionizable groups of the enzyme active site. The results obtained are consistent with the requirement for three groups utilized by urease with pK(a)s equal to 5.3+/-0.2, 6.6+/-0.2 and 9.1+/-0.4. Based on the appearance of the ionization step with pK(a)=5.3 in v(max)-pH, K(M)-pH and K(i)-pH profiles, we assigned this group as participating both in the substrate binding and catalytic reaction. As shown by its presence in v(max)-pH and K(M)-pH curves, the obvious role of the group with pK(a)=9.1 is the participation in the catalytic reaction. One function of the group featuring pK(a)=6.6, which was derived from a two-maxima v(max)-pH profile obtained upon increasing phosphate buffer concentration, an effect the first time observed for urease-phosphate systems, is the substrate binding, another possible function being modulation of the active site structure controlled by the ionic strength. It is also possible that the pK(a)=6.6 is a merger of two pK(a)s close in value. The study establishes that regular bell-shaped activity-pH profiles, commonly reported for urease, entail more complex pH-dependent behavior of the urease active site ionizable groups, which could be experimentally derived using species interacting with the enzyme, in addition to changing solution pH and ionic strength.     

Chemical modification of chalcone isomerase by mercurials and tetrathionate. Evidence for a single cysteine residue in the active site

Chalcone isomerase from soybean is inactivated by stoichiometric amounts of p-mercuribenzoate or HgCl2. Spectral titration of the enzyme with p-mercuribenzoate indicates that a single thiol group is modified. Treatment of modified enzyme with KCN or thiols results in a complete restoration of enzyme activity demonstrating that the inactivation is not due to irreversible protein denaturation. A product of the enzymatic reaction, naringenin, provides complete kinetic protection against inactivation by both mercurials. The binding constant (33 microM) for naringenin determined from the concentration dependence of the protection agrees with the inhibition constant (34 microM) for naringenin as a competitive inhibitor of the catalytic reaction. This agreement demonstrates that the observed kinetic protection results from the specific binding of naringenin to the active site. Incubation of native chalcone isomerase with sodium tetrathionate (0.1 M) results in a slow time-dependent loss of enzymatic activity. The inactivation of chalcone isomerase by tetrathionate and N-ethylmaleimide becomes very rapid in the presence of 6 M urea, indicating that the native tertiary structure is responsible for the low reactivity of the enzymatic thiol. The stoichiometric modification of reduced and denatured chalcone isomerase by [3H] N-ethylmaleimide indicates that the enzyme contains only a single cysteine residue and does not contain any disulfides. The evidence presented suggests that the only half-cystine residue in chalcone isomerase is located in the active site and thereby provides the first clue to the location of the active site in chalcone isomerase.     

Inhibition of urease by bismuth(III): Implications for the mechanism of action of bismuth drugs

Bismuth compounds are widely used for the treatment of peptic ulcers and Helicobacter pylori infections. It has been suggested that enzyme inhibition plays an important role in the antibacterial activity of bismuth towards this bacterium. Urease, an enzyme that converts urea into ammonia and carbonic acid, is crucial for colonization of the acidic environment of the stomach by H. pylori. Here, we show that three bismuth complexes exhibit distinct mechanisms of urease inhibition, with some differences dependent on the source of the enzyme. Bi(EDTA) and Bi(Cys)₃ are competitive inhibitors of jack bean urease with K _i values of 1.74 ± 0.14 and 1.84 ± 0.15 mM, while the anti-ulcer drug, ranitidine bismuth citrate (RBC) is a non-competitive inhibitor with a K _i value of 1.17 ± 0.09 mM. A ¹³C NMR study showed that Bi(Cys)₃ reacts with jack bean urease during a 30 min incubation, releasing free cysteines from the metal complex. Upon incubation with Bi(EDTA) and RBC, the number of accessible cysteine residues in the homohexameric plant enzyme decreased by 5.80 ± 0.17 and 11.94 ± 0.13, respectively, after 3 h of reaction with dithiobis(2-nitrobenzoic acid). Kinetic analysis showed that Bi(EDTA) is both a competitive inhibitor and a time-dependent inactivator of the recombinant Klebsiella aerogenes urease. The active C319A mutant of the bacterial enzyme displays a significantly reduced sensitivity toward inactivation by Bi(EDTA) compared with the wild-type enzyme, consistent with binding of Bi³⁺ to the active site cysteine (Cys³¹⁹) as the mechanism of enzyme inactivation.     

The structural basis of substrate activation in yeast pyruvate decarboxylase. A crystallographic and kinetic study.

The crystal structure of the complex of the thiamine diphosphate dependent tetrameric enzyme pyruvate decarboxylase (PDC) from brewer's yeast strain with the activator pyruvamide has been determined to 2.4 A resolution. The asymmetric unit of the crystal contains two subunits, and the tetrameric molecule is generated by crystallographic symmetry. Structure analysis revealed conformational nonequivalence of the active sites. One of the two active sites in the asymmetric unit was found in an open conformation, with two active site loop regions (residues 104-113 and 290-304) disordered. In the other subunit, these loop regions are well-ordered and shield the active site from the bulk solution. In the closed enzyme subunit, one molecule of pyruvamide is bound in the active site channel, and is located in the vicinity of the thiazolium ring of the cofactor. A second pyruvamide binding site was found at the interface between the Pyr and the R domains of the subunit in the closed conformation, about 10 A away from residue C221. This second pyruvamide molecule might function in stabilizing the unique orientation of the R domain in this subunit which in turn is important for dimer-dimer interactions in the activated tetramer. No difference electron density in the close vicinity of the side chain of residue C221 was found, indicating that this residue does not form a covalent adduct with an activator molecule. Kinetic experiments showed that substrate activation was not affected by oxidation of cysteine residues and therefore does not seem to be dependent on intact thiol groups in the enzyme. The results suggest that a disorder-order transition of two active-site loop regions is a key event in the activation process triggered by the activator pyruvamide and that covalent modification of C221 is not required for this transition to occur. Based on these findings, a possible mechanism for the activation of PDC by its substrate, pyruvate, is proposed.