Inhibition of Chymotrypsin by a Complex of Ortho-Vanadate and Benzohydroxamic Acid:  Structure of the Inert Complex and Its Mechanistic Interpretation

Abstract

Serine proteases, like serine beta-lactamases, are rapidly and covalently inhibited by suitably designed phosph(on)ates. The active sites of these enzymes must, therefore, be able to stabilize the pentacoordinated transition states of phosphyl transfer reactions as well as the tetrahedral transition states of acyl transfers. It follows that these enzymes should also be inhibited by molecules capable of generating inert pentacoordinated species. We (J.H.B. and R.F.P.) have previously shown that these enzymes are, in fact, rapidly and reversibly inhibited by 1:1 complexes of vanadate and hydroxamic acids. In this paper, we present the first crystal structure of an acyl transferase inhibited by vanadate. The complex of vanadate and benzohydroxamic acid is a competitive inhibitor of alpha-chymotrypsin with a KI value of 16 muM. In the structure, obtained at a resolution of 1.5 A, the protein is conformationally little different from the apoenzyme. The vanadium, in a distorted octahedral ligand field, is covalently bound to the active site serine oxygen group. One oxgen ligand, presumably anionic, is located in the oxyanion hole. Another is directed roughly in the direction of the acyl transfer leaving group, and a third in the direction of the S2 site. The hydroxamate is bound to vanadium through the hydroxyl oxygen and also, more weakly, through the carbonyl group, to form a five-membered chelate ring. The effect of this chelation is to place the phenyl group of the inhibitor into the important S1 specificity site. The hydroxamate oxygen is directed in line away from the Ser195 Ogamma, approximating the direction of departure of a leaving group in phosphyl transfer. The entire complex can be seen as a reasonable mimic of a phosphyl transfer transition state where the leaving group is extended into the S1 site.

Vanadium and proteins: Uptake, transport, structure, activity and function

“…The localization of the phenyl ring of the hydroxamate in the S1 binding pocket and the hydroxamate oxygen is discussed by the authors. Globally, the study concludes that chymotrypsin and vanadate(V) are good models for studies involving protease inhibitors due to their ability to unravel the structural details of the catalytic transition states [450].…”

“…25). The V-atom presents a distorted octahedral geometry with six ligands: the mentioned serine, the carbonyl and hydroxyl of the benzohydroxamic acid and three O-atoms, one of which is hydrogen bonded to the backbone N-atom of Gly193 and Ser195 residues [450]. The localization of the phenyl ring of the hydroxamate in the S1 binding pocket and the hydroxamate oxygen is discussed by the authors.…”

“…In fact, a 1.5 Å resolution structure of bovine chymotrypsin, a serine protease, complexed with vanadate(V) and benzohydroxamic acid (PDB: 2P8O) was reported [450] corroborating the previous findings on the inhibition of these proteins by 1:1 complexes of vanadate(V) and hydroxamic acids [451]. The complex formed by vanadate(V) and benzohydroxamic acid is in the active center and the V-atom is covalently bound to the O-atom of Ser195, one of the residues of the so-called catalytic triad, along with His57 and Asp102 (Fig.…”

A preliminary neutron diffraction study of γ-chymotrypsin

“…Crystals of -chymotrypsin failed to form using previously published conditions (Stoddard et al, 1990). Further screening using Additive Screen (Hampton Research, Aliso Viejo, California, USA) demonstrated that supplementation of the previously reported condition (Stoddard et al, 1990) with sodium iodide was sufficient to produce large crystals of -chymotrypsin (Moulin et al, 2007). Crystal size was further increased using a modified sitting-drop technique in which an uncapped 0.5 dram vial was nested in a tightly sealed scintillation vial.…”

Spectroscopic and theoretical study on the interaction between diperoxovanadate complexes and glycyl-histidine

“…The systems of NH 4 VO 3 /H 2 O 2 /histidine-like ligands as models to imitate the active site of haloperoxidases have been explored by Conte and co-workers [14,16]. Pratt and co-workers investigated the formation and structures of complexes between vanadates and hydroxamic acids at neutral pH, and their mechanism of inhibition of ␤-lactamase [17][18][19]. Although much effort has been expended on investigating the complicated structures and chemical behaviors of diperoxovanadates in solution, there are still problems not completely elucidated owing to the complexity of vanadium chemistry.…”