Protein allostery is based on the existence of multiple conformations in equilibrium linked to distinct functional properties. Although evidence of allosteric transitions is relatively easy to identify by functional studies, structural detection of a pre-existing equilibrium between alternative conformations remains challenging even for textbook examples of allosteric proteins. Kinetic studies show that the trypsin-like protease thrombin exists in equilibrium between two conformations where the active site is either collapsed (E*) or accessible to substrate (E). However, structural demonstration that the two conformations exist in the same enzyme construct free of ligands has remained elusive. Here we report the crystal structure of the thrombin mutant N143P in the E form, which complements the recently reported structure in the E* form, and both the E and E* forms of the thrombin mutant Y225P. The side chain of W215 moves 10.9 Å between the two forms, causing a displacement of 6.6 Å of the entire 215–217 segment into the active site that in turn opens or closes access to the primary specificity pocket. Rapid kinetic measurements of p-aminobenzamidine binding to the active site confirm the existence of the E*-E equilibrium in solution for wild-type and the mutants N143P and Y225P. These findings provide unequivocal proof of the allosteric nature of thrombin and lend strong support to the recent proposal that the E*-E equilibrium is a key property of the trypsin fold.

Oxalate secretion by fungi is known to be associated with fungal pathogenesis. In addition, oxalate toxicity is a concern for the commercial application of fungi in the food and drug industries. Although oxalate is generated through several different biochemical pathways, oxaloacetate acetylhydrolase (OAH)-catalyzed hydrolytic cleavage of oxaloacetate appears to be an especially important route. Below, we report the cloning of the Botrytis cinerea oahA gene and the demonstration that the disruption of this gene results in the loss of oxalate formation. In addition, through complementation we have shown that the intact B. cinerea oahA gene restores oxalate production in an Aspergillus niger mutant strain, lacking a functional oahA gene. These observations clearly indicate that oxalate production in A. niger and B. cinerea is solely dependent on the hydrolytic cleavage of oxaloacetate catalyzed by OAH. In addition, the B. cinera oahA gene was overexpressed in Escherichia coli and the purified OAH was used to define catalytic efficiency, substrate specificity, and metal ion activation. These results are reported along with the discovery of the mechanism-based, tight binding OAH inhibitor 3,3-difluorooxaloacetate (K i ‫؍‬ 68 nM). Finally, we propose that cellular uptake of this inhibitor could reduce oxalate production.Numerous filamentous fungi, including the food biotechnology fungus Aspergillus niger, the opportunistic human pathogen Aspergillus fumigatus, the phytopathogenic fungi Botrytis cinerea and Sclerotinia sclerotiorum, as well as many brown-rot and white-rot basidiomycetes, are able to efficiently produce large quantities of oxalate (1, 2). It is known that oxalate secretion is associated with fungal pathogenesis (1, 3-6). In the wood-rotting fungus Fomitopsis palustris oxalate is formed as the product of glucose metabolism (7). We recently initiated investigations of the oxalate biosynthetic pathway to develop a genomic-based method for distinguishing between oxalate producing and non-producing fungi. An additional goal of this effort was to identify enzyme inhibitors that could be used to arrest oxalate formation in targeted fungi.To attenuate oxalate production in fungi, it is necessary to first identify the major pathway responsible for oxalate formation. There are three potential routes for production of oxalate in fungi: oxidation of glyoxylate (8, 9), oxidation of glycolaldehyde (10), and hydrolysis of oxaloacetate (11). The results of studies of [ 14 C]CO 2 incorporation into the metabolite pools of A. niger indicate that oxalate is derived from oxaloacetate (12). This finding parallels the results of earlier work on the purification of an enzyme "oxalacetalase" (now known as oxaloacetate acetylhydrolase or OAH) 4 that catalyzes the hydrolytic cleavage of oxaloacetate to form acetate and oxalate (11). In a subsequent study, a mutant A. niger strain, NW228 (13), was found to be deficient in both oxalate production and in the synthesis of active OAH (14). These observations suggest that oxalate is ...

The molecular mechanism of thrombin activation by Na ؉ remains elusive. Its kinetic formulation requires extension of the classical Botts-Morales theory for the action of a modifier on an enzyme to correctly account for the contribution of the E*, E, and E:Na ؉ forms. peptide bond, which is likely flipped in the absence of cation. Absolute conservation of the 143-192 H-bond in trypsin-like proteases and the importance of the oxyanion hole in protease function suggest that this mechanism of Na؉ activation is present in all Na ؉ -activated trypsin-like proteases.Regulation of activity through metal ion complexation plays a key role in many enzyme-catalyzed reactions, and over onethird of known proteins are metalloproteins (1-3). The earliest evidence for monovalent cation (M ϩ ) activation of enzymes was provided by Boyer (4) with the discovery of the absolute requirement of K ϩ by pyruvate kinase (5). Monod demonstrated Na ϩ -dependent catalytic rate enhancement in ␤-galactosidase (6). Following these discoveries, many enzymes were observed to display increased activity in the presence of M ϩ (7). A recent classification of M ϩ

Background: The x-ray structure and mechanism of activation of prothrombin remain elusive. Results: X-ray and solution studies document conformation flexibility of prothrombin. The two sites of cleavage at Arg-271 and Arg-320 have distinct solvent accessibility. Conclusion: Burial of Arg-320 prevents prothrombin autoactivation and directs prothrombinase to cleave at Arg-271 first. Significance: A structure-based mechanism of prothrombin activation emerges.

Background: Thrombin is generated from zymogen precursors with the assistance of cofactors. Results: Thrombin precursors prethrombin-2 and prothrombin are capable of catalytic activity and autoactivate. Conclusion: Conformational selection regulates activity in the mature protease and has the potential to unleash autoactivation in the zymogen. Significance: The paradigm of the inactive zymogen to active protease conversion needs revision to account for conformational selection.

Phosphonopyruvate (P-pyr) hydrolase (PPH), a member of the phosphoenolpyruvate (PEP) mutase/isocitrate lyase (PEPM/ICL) superfamily, hydrolyzes P-pyr and shares the highest sequence identity and functional similarity with PEPM. Recombinant PPH from Variovorax sp. Pal2 was expressed in Escherichia coli and purified to homogeneity. Analytical gel filtration indicated that the protein exists in solution predominantly as a tetramer. The PPH pH rate profile indicates maximal activity over a broad pH range. The steady-state kinetic constants determined for a rapid equilibrium ordered kinetic mechanism with Mg2+ binding first (Kd = 140 +/- 40 microM), are kcat = 105 +/- 2 s(-1) and P-pyr Km = 5 +/- 1 microM. PEP (slow substrate kcat = 2 x 10(-4) s(-1)), oxalate, and sulfopyruvate are competitive inhibitors with Ki values of 2.0 +/- 0.1 mM, 17 +/- 1 microM, and 210 +/- 10 microM, respectively. Three PPH crystal structures have been determined, that of a ligand-free enzyme, the enzyme bound to Mg2+ and oxalate (inhibitor), and the enzyme bound to Mg2+ and P-pyr (substrate). The complex with the inhibitor was obtained by cocrystallization, whereas that with the substrate was obtained by briefly soaking crystals of the ligand-free enzyme with P-pyr prior to flash cooling. The PPH structure resembles that of the other members of the PEPM/ICL superfamily and is most similar to the functionally related enzyme, PEPM. Each monomer of the dimer of dimers exhibits an (alpha/beta)8 barrel fold with the eighth helix swapped between two molecules of the dimer. Both P-pyr and oxalate are anchored to the active site by Mg2+. The loop capping the active site is disordered in all three structures, in contrast to PEPM, where the equivalent loop adopts an open or disordered conformation in the unbound state but sequesters the inhibitor from solvent in the bound state. Crystal packing may have favored the open conformation of PPH even when the enzyme was cocrystallized with the oxalate inhibitor. Structure alignment of PPH with other superfamily members revealed two pairs of invariant or conservatively replaced residues that anchor the flexible gating loop. The proposed PPH catalytic mechanism is analogous to that of PEPM but includes activation of a water nucleophile with the loop Thr118 residue.

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Aspergillus niger produces oxalic acid through the hydrolysis of oxaloacetate, catalyzed by the cytoplasmic enzyme oxaloacetate acetylhydrolase (OAH). The A. niger genome encodes four additional open reading frames with strong sequence similarity to OAH yet only the oahA gene encodes OAH activity. OAH and OAH-like proteins form subclass of the isocitrate lyase/PEP mutase enzyme superfamily, which is ubiquitous present filamentous fungi. Analysis of function-specific residues using a superfamily-based approach revealed an active site serine as a possible sequence marker for OAH activity. We propose that presence of this serine in family members correlates with presence of OAH activity whereas its absence correlates with absence of OAH. This hypothesis was tested by carrying out a serine mutagenesis study with the OAH from the fungal oxalic acid producer Botrytis cinerea and the OAH active plant petal death protein as test systems.