Molecular modeling reveals the possible importance of a carbonyl oxygen binding pocket for the catalytic mechanism of p-hydroxybenzoate hydroxylase

p-Hydroxybenzoate hydroxylase catalyzes the hydroxylation of an aromatic substrate and uses flavin as a cofactor. The reaction probably occurs via a flavin 4a-hydroperoxide intermediate. In this study the crystal structure of 4a,5-epoxyethano-3-methyl-4a,5-dihydrolumiflavin, an analogue of the flavin 4a-hydroperoxide intermediate, was fitted to the active site in the crystal structure of the p-hydroxybenzoate hydroxylase-3,4-dihydroxybenzoate complex. This model of an important catalytic intermediate fitted very well in the active site of p-hydroxybenzoate hydroxylase. The most striking result was that whereas with the normal flavin, the 0-4 of the flavin ring makes only poor hydrogen bonds with the protein, with the flavin 4a-hydroperoxide analogue, the same 0-4 makes strong hydrogen bonds with the NH groups of Gly-46 and Val-47. These two NH groups form a carbonyl oxygen binding pocket which has a geometry almost identical to the oxyanion hole found in several proteases. The possible consequences of this model for the reaction mechanism of p-hydroxybenzoate hydroxylase are discussed.     

Characterization of the conformational changes of acetohydroxy acid isomeroreductase induced by the binding of Mg2+ ions, NADPH, and a competitive inhibitor.

Acetohydroxy acid isomeroreductase (EC 1.1.1.86), the second enzyme of the parallel branched chain amino acid pathway, is a homodimer with an Mr of approximately 114000 which in the presence of Mg2+ ions catalyzes an unusual alkyl migration followed by an NADPH-dependent reduction. Prior binding of NADPH and Mg2+ to the enzyme was shown to be required for substrate or competitive inhibitor [N-hydroxy-N-isopropyloxamate (IpOHA)] binding [Dumas, R., et al. (1994) Biochem. J. 301, 813-820]. Moreover, crystallographic data for the enzyme-NADPH-Mg2+-IpOHA complex [Biou, V., et al. (1997) EMBO J. 16, 3405-3415] have shown that IpOHA was completely buried inside the active site. These observations raised the question of how the reaction intermediate analogue inhibitor can reach the active site and implied that conformational changes occurred during the binding process. With a view of characterizing these conformational changes, H-D exchange experiments combined with mass spectrometry were performed. Results demonstrated that Mg2+ ions and NADPH binding led to an initial conformational change at the interface of the two domains of each monomer. Binding of the two cofactors to isomeroreductase alters the structure of the active site to promote inhibitor (substrate) binding, in agreement with the ordered mechanism of the enzyme. Structural changes remote from the active site were also found. They were interpreted as long-range structural effects on the two domains and on the two monomers in the time course of the ligand binding process.     

Structure of a pancreatic alpha-amylase bound to a substrate analogue at 2.03 A resolution.

The structure of pig pancreatic alpha-amylase in complex with carbohydrate inhibitor and proteinaceous inhibitors is known but the successive events occurring at the catalytic center still remain to be elucidated. The X-ray structure analysis of a crystal of pig pancreatic alpha-amylase (PPA, EC 3.2.1.1.) soaked with an enzyme-resistant substrate analogue, methyl 4,4'-dithio-alpha-maltotrioside, showed electron density corresponding to the binding of substrate analogue molecules at the active site and at the "second binding site." The electron density observed at the active site was interpreted in terms of overlapping networks of oligosaccharides, which show binding of substrate analogue molecules at subsites prior to and subsequent to the cleavage site. A weaker patch of density observed at subsite -1 (using a nomenclature where the site of hydrolysis is taken to be between subsites -1 and +1) was modeled with water molecules. Conformational changes take place upon substrate analogue binding and the "flexible loop" that constitutes the surface edge of the active site is observed in a specific conformation. This confirms that this loop plays an important role in the recognition and binding of the ligand. The crystal structure was refined at 2.03 A resolution, to an R-factor of 16.0 (Rfree, 18.5).     

The crystal structure of phosphinothricin in the active site of glutamine synthetase illuminates the mechanism of enzymatic inhibition

Phosphinothricin is a potent inhibitor of the enzyme glutamine synthetase (GS). The resolution of the native structure of GS from Salmonella typhimurium has been extended to 2.5 A resolution, and the improved model is used to determine the structure of phosphinothricin complexed to GS by difference Fourier methods. The structure suggests a noncovalent, dead-end mechanism of inhibition. Phosphinothricin occupies the glutamate substrate pocket and stabilizes the Glu327 flap in a position which blocks the glutamate entrance to the active site, trapping the inhibitor on the enzyme. One oxygen of the phosphinyl group of phosphinothricin appears to be protonated, because of its proximity to the carboxylate group of Glu327. The other phosphinyl oxygen protrudes into the negatively charged binding pocket for the substrate ammonium, disrupting that pocket. The distribution of charges in the glutamate binding pocket is complementary to those of phosphinothricin. The presence of a second ammonium binding site within the active site is confirmed by its analogue thallous ion, marking the ammonium site and its protein ligands. The inhibition of GS by methionine sulfoximine can be explained by the same mechanism. These models of inhibited GS further illuminate its catalytic mechanism.     

Using substrate analogues to probe the kinetic mechanism and active site of Escherichia coli MenD

MenD catalyzes the thiamin diphosphate-dependent decarboxylative carboligation of α-ketoglutarate and isochorismate. The enzyme is essential for menaquinone biosynthesis in many bacteria and has been proposed to be an antibiotic target. The kinetic mechanism of this enzyme has not previously been demonstrated because of the limitations of the UV-based kinetic assay. We have reported the synthesis of an isochorismate analogue that acts as a substrate for MenD. The apparent weaker binding of this analogue is advantageous in that it allows accurate kinetic experiments at substrate concentrations near K(m). Using this substrate in concert with the dead-end inhibitor methyl succinylphosphonate, an analogue of α-ketoglutarate, we show that MenD follows a ping-pong kinetic mechanism. Using both the natural and synthetic substrates, we have measured the effects of 12 mutations of residues at the active site. The results give experimental support to previous models and hypotheses and allow observations unavailable using only the natural substrate.     

Active site analysis of the potential antimicrobial target aspartate semialdehyde dehydrogenase.

Aspartate-beta-semialdehyde dehydrogenase (ASADH) lies at the first branch point in the biosynthetic pathway through which bacteria, fungi, and the higher plants synthesize amino acids, including lysine and methionine and the cell wall component diaminopimelate from aspartate. Blocks in this biosynthetic pathway, which is absent in mammals, are lethal, and inhibitors of ASADH may therefore serve as useful antibacterial, fungicidal, or herbicidal agents. We have determined the structure of ASADH from Escherichia coli by crystallography in the presence of its coenzyme and a substrate analogue that acts as a covalent inhibitor. This structure is comparable to that of the covalent intermediate that forms during the reaction catalyzed by ASADH. The key catalytic residues are confirmed as cysteine 135, which is covalently linked to the intermediate during the reaction, and histidine 274, which acts as an acid/base catalyst. The substrate and coenzyme binding residues are also identified, and these active site residues are conserved throughout all of the ASADH sequences. Comparison of the previously determined apo-enzyme structure [Hadfield et al. J. Mol. Biol. (1999) 289, 991-1002] and the complex presented here reveals a conformational change that occurs on binding of NADP that creates a binding site for the amino acid substrate. These results provide a structural explanation for the preferred order of substrate binding that is observed kinetically.     

Molecular mechanism of allosteric substrate activation in a thiamine diphosphate-dependent decarboxylase

Thiamine diphosphate-dependent enzymes are involved in a wide variety of metabolic pathways. The molecular mechanism behind active site communication and substrate activation, observed in some of these enzymes, has since long been an area of debate. Here, we report the crystal structures of a phenylpyruvate decarboxylase in complex with its substrates and a covalent reaction intermediate analogue. These structures reveal the regulatory site and unveil the mechanism of allosteric substrate activation. This signal transduction relies on quaternary structure reorganizations, domain rotations, and a pathway of local conformational changes that are relayed from the regulatory site to the active site. The current findings thus uncover the molecular mechanism by which the binding of a substrate in the regulatory site is linked to the mounting of the catalytic machinery in the active site in this thiamine diphosphate-dependent enzyme.     

Structure of aspartate racemase complexed with a dual substrate analogue, citric acid, and implications for the reaction mechanism

Pyrococcus horikoshii OT3 aspartate racemase (PhAspR) catalyzes the interconversion between L- and D-aspartate. The X-ray structure of PhAspR revealed a pseudo mirror-symmetric distribution of the residues around its active site, which is very reasonable for its chiral substrates, L-aspartate and D-aspartate. In this study, we have determined the crystal structure of an inactive mutant PhAspR complexed with a citric acid (Cit) at a resolution of 2.0 A. Cit contains the substrate analogue moieties of both L- and D-aspartate and exhibits a low competitive inhibition activity against PhAspR. In the structure, Cit binds to the catalytic site of PhAspR, which induced the conformational change to close the active site. The distance between the thiolates was estimated to be 7.4 A, representing a catalytic state and the substrate binding modes of PhAspR. Two conserved basic residues, Arg48 and Lys164, seem to be indispensable for PhAspR activity. Arg48 is thought to be responsible for recognizing carboxyl groups of the substrates L-/D-aspartates and stabilizing a reaction intermediate, and Lys164 is responsible for stabilizing a closed state structure. In this structure, the L-aspartate moiety of Cit is likely to take the substrate position of the PhAspR-substrate complex, which is very similar to that of Glutamate racemase. There is also another possibility that the two substrate analogue moieties of the bound Cit reflect the binding modes of both L- and D-aspartates. Based on the PhAspR-Cit complex structure, the reaction mechanism of aspartate racemase was elucidated.     

On the multiple functional roles of the active site histidine in catalysis and allosteric regulation of Escherichia coli glucosamine 6-phosphate deaminase

The active site of glucosamine-6-phosphate deaminase (EC 3.5.99.6, formerly 5.3.1.10) from Escherichia coli was first characterized on the basis of the crystallographic structure of the enzyme bound to the competitive inhibitor 2-amino-2-deoxy-glucitol 6-phosphate. The structure corresponds to the R allosteric state of the enzyme; it shows the side-chain of His143 in close proximity to the O5 atom of the inhibitor. This arrangement suggests that His143 could have a role in the catalysis of the ring-opening step of glucosamine 6-phosphate whose alpha-anomer is the true substrate. The imidazole group of this active-site histidine contacts the carboxy groups from Glu148 and Asp141, via its Ndelta1 atom [Oliva et al. (1995) Structure 3, 1323-1332]. These interactions change in the T state because the side chain of Glu148 moves toward the allosteric site, leaving at the active site the dyad Asp141-His143 [Horjales et al. (1999) Structure 7, 527-536]. In this research, a dual approach using site-directed mutagenesis and controlled chemical modification of histidine residues has been used to investigate the role of the active-site histidine. Our results support a multifunctional role of His143; in the forward reaction, it is involved in the catalysis of the ring-opening step of the substrate, glucosamine 6-P. In the reverse reaction, the substrate fructose 6-P binds in its open chain, carbonylic form. The role of His143 in the binding of both glucosamine 6-P and reaction intermediates in their extended-chain forms was demonstrated by binding experiments using the reaction intermediate analogue, 2-amino-2-deoxy-D-glucitol 6-phosphate. His143 was also shown to be a critical residue for the conformational coupling between active and allosteric sites. From the pH dependence of the reactivity of the active site histidine to diethyl dicarbonate, we observed a pK(a) change of 1.2 units to the acid side when the enzyme undergoes the allosteric T to R transition during which the side chain of Glu148 moves toward the active site. The kinetic study of the Glu148-Gln mutant deaminase shows that the loss of the carboxy group and its replacement with the corresponding amide modifies the k(cat) versus pH profile of the enzyme, suggesting that the catalytic step requiring the participation of His143 has become rate-limiting. This, in turn, indicates that the interaction Glu148-His143 in the wild-type enzyme in the R state contributes to make the enzyme functional over a wide pH range.     

Mechanistic inferences from the binding of ligands to LpxC, a metal-dependent deacetylase

The metal-dependent deacetylase LpxC catalyzes the first committed step of lipid A biosynthesis in Gram-negative bacteria. Accordingly, LpxC is an attractive target for the development of inhibitors that may serve as potential new antibiotics for the treatment of Gram-negative bacterial infections. Here, we report the 2.7 A resolution X-ray crystal structure of LpxC complexed with the substrate analogue inhibitor TU-514 and the 2.0 A resolution structure of LpxC complexed with imidazole. The X-ray crystal structure of LpxC complexed with TU-514 allows for a detailed examination of the coordination geometry of the catalytic zinc ion and other enzyme-inhibitor interactions in the active site. The hydroxamate group of TU-514 forms a bidentate chelate complex with the zinc ion and makes hydrogen bond interactions with conserved active site residues E78, H265, and T191. The inhibitor C-4 hydroxyl group makes direct hydrogen bond interactions with E197 and H58. Finally, the C-3 myristate moiety of the inhibitor binds in the hydrophobic tunnel of the active site. These intermolecular interactions provide a foundation for understanding structural aspects of enzyme-substrate and enzyme-inhibitor affinity. Comparison of the TU-514 complex with cacodylate and imidazole complexes suggests a possible substrate diphosphate binding site and highlights residues that may stabilize the tetrahedral intermediate and its flanking transition states in catalysis. Evidence of a catalytic zinc ion in the native zinc enzyme coordinated by H79, H238, D242, and two water molecules with square pyramidal geometry is also presented. These results suggest that the native state of this metallohydrolase may contain a pentacoordinate zinc ion, which contrasts with the native states of archetypical zinc hydrolases such as thermolysin and carboxypeptidase A.     

Purification, Characterization, and Localization of Aspartoacylase from Bovine Brain

Canavan disease, an autosomal recessive disorder, is characterized biochemically by N-acetylaspartic aciduria and aspartoacylase (N-acyl-L-aspartate amidohydrolase; EC 3.5.1.15) deficiency. However, the role of aspartoacylase and N-acetylaspartic acid in brain metabolism is unknown. Aspartoacylase has been purified to apparent homogeneity with a specific activity of approximately 19,000-20,000 nmol of aspartate released/mg of protein. The native enzyme is a 58-kDa monomer. The purified aspartoacylase activity is enhanced by divalent cations, nonionic detergents, and dithiothreitol. Low levels of dithiothreitol or beta-mercaptoethanol are required for enzyme stability. Aspartoacylase has a Km of 8.5 x 10(-4) M and a Vmax of 43,000 nmol/min/mg of protein. Inhibition of aspartoacylase by glycyl-L-aspartate and amino derivatives of D-aspartic acid suggests that the carbon backbone of the substrate is primarily involved in its interaction with the active site and that a blocked amino group is essential for the catalytic activity of aspartoacylase. Biochemical and immunocytochemical studies revealed that aspartoacylase is localized to white matter, whereas the N-acetylaspartic acid concentration is threefold higher in gray matter than in white matter. Our studies so far indicate that aspartoacylase is conserved across species during evolution and suggest a significant role for aspartoacylase and N-acetylaspartic acid in normal brain biology.     

High resolution reaction intermediates of rabbit muscle fructose-1,6-bisphosphate aldolase: substrate cleavage and induced fit

Crystal structures were determined to 1.8 A resolution of the glycolytic enzyme fructose-1,6-bis(phosphate) aldolase trapped in complex with its substrate and a competitive inhibitor, mannitol-1,6-bis(phosphate). The enzyme substrate complex corresponded to the postulated Schiff base intermediate and has reaction geometry consistent with incipient C3-C4 bond cleavage catalyzed Glu-187, which is adjacent by to the Schiff base forming Lys-229. Atom arrangement about the cleaved bond in the reaction intermediate mimics a pericyclic transition state occurring in nonenzymatic aldol condensations. Lys-146 hydrogen-bonds the substrate C4 hydroxyl and assists substrate cleavage by stabilizing the developing negative charge on the C4 hydroxyl during proton abstraction. Mannitol-1,6-bis(phosphate) forms a noncovalent complex in the active site whose binding geometry mimics the covalent carbinolamine precursor. Glu-187 hydrogen-bonds the C2 hydroxyl of the inhibitor in the enzyme complex, substantiating a proton transfer role by Glu-187 in catalyzing the conversion of the carbinolamine intermediate to Schiff base. Modeling of the acyclic substrate configuration into the active site shows Glu-187, in acid form, hydrogen-bonding both substrate C2 carbonyl and C4 hydroxyl, thereby aligning the substrate ketose for nucleophilic attack by Lys-229. The multifunctional role of Glu-187 epitomizes a canonical mechanistic feature conserved in Schiff base-forming aldolases catalyzing carbohydrate metabolism. Trapping of tagatose-1,6-bis(phosphate), a diastereoisomer of fructose 1,6-bis(phosphate), displayed stereospecific discrimination and reduced ketohexose binding specificity. Each ligand induces homologous conformational changes in two adjacent alpha-helical regions that promote phosphate binding in the active site.     

Crystal structure and mechanism of tryptophan 2,3-dioxygenase, a heme enzyme involved in tryptophan catabolism and in quinolinate biosynthesis

The structure of tryptophan 2,3-dioxygenase (TDO) from Ralstonia metallidurans was determined at 2.4 A. TDO catalyzes the irreversible oxidation of l-tryptophan to N-formyl kynurenine, which is the initial step in tryptophan catabolism. TDO is a heme-containing enzyme and is highly specific for its substrate l-tryptophan. The structure is a tetramer with a heme cofactor bound at each active site. The monomeric fold, as well as the heme binding site, is similar to that of the large domain of indoleamine 2,3-dioxygenase, an enzyme that catalyzes the same reaction except with a broader substrate tolerance. Modeling of the putative (S)-tryptophan hydroperoxide intermediate into the active site, as well as substrate analogue and mutagenesis studies, are consistent with a Criegee mechanism for the reaction.     

High-resolution X-ray diffraction study of the complex between endothiapepsin and an oligopeptide inhibitor: the analysis of the inhibitor binding and description of the rigid body shift in the enzyme.

The conformation of the synthetic renin inhibitor CP-69,799, bound to the active site of the fungal aspartic proteinase endothiapepsin (EC 3.4.23.6), has been determined by X-ray diffraction at 1.8 A resolution and refined to the crystallographic R factor of 16%. CP-69,799 is an oligopeptide transition--state analogue inhibitor that contains a new dipeptide isostere at the P1-P1' position. This dipeptide isostere is a nitrogen analogue of the well-explored hydroxyethylene dipeptide isostere, wherein the tetrahedral P1' C alpha atom has been replaced by trigonal nitrogen. The inhibitor binds in the extended conformation, filling S4 to S3' pockets, with hydroxyl group of the P1 residue positioned symmetrically between the two catalytic aspartates of the enzyme. Interactions between the inhibitor and the enzyme include 12 hydrogen bonds and extensive van der Waals contacts in all the pockets, except for S3'. The crystal structure reveals a bifurcated orientation of the P2 histidine side chain and an interesting relative rotation of the P3 phenyl ring to accommodate the cyclohexyl side chain at P1. The binding of the inhibitor to the enzyme, while producing no large distortions in the enzyme active site cleft, results in small but significant change in the relative orientation of the two endothiapepsin domains. This structural change may represent the action effected by the proteinase as it distorts its substrate towards the transition state for proteolytic cleavage.     

Spectroscopic studies of inhibited alcohol dehydrogenase from Thermoanaerobacter brockii: proposed structure for the catalytic intermediate state

Thermoanaerobacter brockii alcohol dehydrogenase (TbADH) catalyzes the reversible oxidation of secondary alcohols to the corresponding ketones using NADP(+) as the cofactor. The active site of the enzyme contains a zinc ion that is tetrahedrally coordinated by four protein residues. The enzymatic reaction leads to the formation of a ternary enzyme-cofactor-substrate complex; and catalytic hydride ion transfer is believed to take place directly between the substrate and cofactor at the ternary complex. Although crystallographic data of TbADH and other alcohol dehydrogenases as well as their complexes are available, their mode of action remains to be determined. It is firmly established that the zinc ion is essential for catalysis. However, there is no clear agreement about the coordination environment of the metal ion and the competent reaction intermediates during catalysis. We used a combination of X-ray absorption, circular dichroism (CD), and fluorescence spectroscopy, together with structural analysis and modeling studies, to investigate the ternary complexes of TbADH that are bound to a transition-state analogue inhibitor. Our structural and spectroscopic studies indicated that the coordination sphere of the catalytic zinc site in TbADH undergoes conformational changes when it binds the inhibitor and forms a pentacoordinated complex at the zinc ion. These studies provide the first active site structure of bacterial ADH bound to a substrate analogue. Here, we suggest the active site structure of the central intermediate complex and, more specifically, propose the substrate-binding site in TbADH.     

Reaction mechanism of glyoxalase I explored by an X-ray crystallographic analysis of the human enzyme in complex with a transition state analogue.

The structures of human glyoxalase I in complexes with S-(N-hydroxy-N-p-iodophenylcarbamoyl)glutathione (HIPC-GSH) and S-p-nitrobenzyloxycarbonylglutathione (NBC-GSH) have been determined at 2.0 and 1.72 A resolution, respectively. HIPC-GSH is a transition state analogue mimicking the enediolate intermediate that forms along the reaction pathway of glyoxalase I. In the structure, the hydroxycarbamoyl function is directly coordinated to the active site zinc ion. In contrast, the equivalent group in the NBC-GSH complex is approximately 6 A from the metal in a conformation that may resemble the product complex with S-D-lactoylglutathione. In this complex, two water molecules occupy the liganding positions at the zinc ion occupied by the hydroxycarbamoyl function in the enediolate analogue complex. Coordination of the transition state analogue to the metal enables a loop to close down over the active site, relative to its position in the product-like structure, allowing the glycine residue of the glutathione moiety to hydrogen bond with the protein. The structure of the complex with the enediolate analogue supports an "inner sphere mechanism" in which the GSH-methylglyoxal thiohemiacetal substrate is converted to product via a cis-enediolate intermediate. The zinc ion is envisioned to play an electrophilic role in catalysis by directly coordinating this intermediate. In addition, the carboxyl of Glu 172 is proposed to be displaced from the inner coordination sphere of the metal ion during substrate binding, thus allowing this group to facilitate proton transfer between the adjacent carbon atoms of the substrate. This proposal is supported by the observation that in the complex with the enediolate analogue the carboxyl group of Glu 172 is 3.3 A from the metal and is in an ideal position for reprotonation of the transition state intermediate. In contrast, Glu 172 is directly coordinated to the zinc ion in the complexes with S-benzylglutathione and with NBC-GSH.     

Structural basis for differences in substrate specificity of aspartate aminotransferase isoenzymes

X-Ray structural data concerning the substrate binding site of cytosolic chicken aspartate aminotransferase (AspAT) are reported. The structure of the complex of AspAT with the substrate-like inhibitor maleate has been refined at 2.2 A resolution. The lengths of hydrogen bonds between a bound molecule of maleate and side chains of amino acid residues in the active site are presented as well as other interatomic distances in the substrate binding site. The data obtained for the cytosolic AspAT have been compared with those for the mitochondrial chicken AspAT. It has been inferred that differences in substrate specificity of the AspAT isoenzymes are determined by interactions involving amino acid residues which are situated in the immediate vicinity of the active site and influence ionization or orientation of functional groups interacting with substrate. An explanation is suggested for different rates of transamination of aromatic amino acids in the active sites of the cytosolic and mitochondrial isoenzymes.     

Studies of the active site of cytochrome P-450scc with a high-affinity spin-labeled inhibitor

The intramolecular site of P-450scc for conversion of cholesterol to pregnenolone involves a substrate site, an active site, and a site for transmission of electrons. The substrate site was studied with a high-affinity, high-potency nitroxide spin-labeled inhibitor of cholesterol side-chain cleavage. This substance, 17 alpha-hydroxy-11-deoxycorticosterone nitroxide (SL-V), has an affinity comparable to that of the most active substrate inhibitors ever reported and 2-50 times greater than that of the natural substrate cholesterol. Competition experiments with cholesterol and its analogues confirmed that SL-V binds reversibly to the substrate site. Titration experiments showed a single binding site on the P-450 molecule. The substrate site is on the apoprotein and has little or no direct interaction with the heme. Spin-spin interactions between the Fe3+ and side-chain or A-ring spin-labeled groups could not be demonstrated, which is consistent with carbons 22 and 20 being closest to the heme iron. We postulate that substrate disrupts a histidine nitrogen coordination with the heme iron and induces conformational changes in the apoprotein. These changes lead to increased affinity for iron-sulfur protein.     

Kinetics and mechanism of refolding of bovine carbonic anhydrase. A probe study of the formation of the active site.

In kinetic studies of the folding of bovine carbonic anhydrase from disorganized to native structure, an azosulfonamide, 2-(4-sulfomylphenylazo)-7-acetamido-1-hydroxynaphthalene-3,6-disulfonate (I), has been used as a probe to follow the dynamics of formation of the active site region. The probe is a specific inhibitor of the native enzyme that binds in the active site crevice. The experiments, with previous data (Yazgan, A., and Henkens, R. W. (1972), Biochemistry 11, 1314), show that a tight binding site for I forms at an intermediate stage in the folding process. A subsequent conformational change perturbs the visible absorption and circular dichroism of bound I and could result in even tighter binding. The subsequent change completes formation of the active site. This is shown by results from separate experiments on the kinetics of recovery of activity (p-nitrophenyl acetate as substrate). Similar probe methods could be used with other proteins and enzymes to study the kinetics and mechanism of regeneration of specific sites--for example, the active site.