A unique "enzyme catalytic promiscuity" has recently been observed, wherein a phosphodiester and a phosphonate ester are hydrolyzed by a dinuclear aminopeptidase and its metal derivatives from Streptomyces griseus (SgAP) [Park, H. I., Ming, L.-J. (1999) Angew. Chem., Int. Ed. Engl. 38, 2914-2916 and Ercan, A., Park, H. I., Ming, L.-J. (2000) Chem. Commun. 2501-2502]. Because tetrahedral phosphocenters often serve as transition-state inhibitors toward the hydrolysis of the peptide, phosphoester hydrolysis by peptidases is thus not expected to occur effectively and must take place through a unique mechanism. Owing to the very different structures and mechanistic requirements between phosphoesters and peptides during hydrolysis, the study of this effective phosphodiester hydrolysis by SgAP may provide further insight into the action of this enzyme that is otherwise not obtainable from regular peptide substrates. We present herein a detailed investigation of both peptide and phosphodiester hydrolyses catalyzed by SgAP. The latter exhibits a first-order rate enhancement of 4 x 10(10)-fold compared to the uncatalyzed reaction at pH 7.0 and 25 degrees C. The results suggest that peptide and phosphodiester hydrolyses by SgAP may share a common reaction mechanism to a certain extent. However, their differences in pH dependence, phosphate and fluoride inhibition patterns, and proton inventory reflect that they must follow different pathways. Mechanisms for the two hydrolyses are drawn on the basis of the results, which provide the foundation for further investigation of the catalytic promiscuity of this enzyme by means of physical and molecular biology methods. The catalytic versatility of SgAP suggests that this enzyme may serve as a unique "natural model system" for further investigation of dinuclear hydrolysis. A better understanding of enzyme catalytic promiscuity is also expected to shed light on the evolution and action of enzymes.

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Serralysin is a bacterial Zn-endopeptidase which has been considered a virulence factor to cause tissue damage and anaphylactic response. It contains a coordinated Tyr that is unique to the astacin-like Zn enzymes. The coordinated Tyr has been proposed to play an important role in the action of this endopeptidase family. Several metal-substituted derivatives of serralysin (including Mn2+, Co2+, Ni2+, Cu2+, and Cd2+ derivatives) are found to exhibit significant activities. Particularly, the Co- and Cu-substituted derivatives exhibit much higher activities than the native serralysin toward the hydrolysis of the tripeptide mimic benzoyl-Arg- p-nitroanilide, i.e., 35 and 49 times higher in k(cat) and 33 and 26 times in k(cat)/ K(m), respectively. Such remarkably higher activities of metal-substituted derivatives, especially the Cu derivative, than that of the native Zn enzyme are rare in the literature, reflecting the uniqueness of this enzyme among all Zn enzymes. The significantly different k(cat) yet similar K(m) values among the several metal derivatives suggests that the metal center is involved in catalysis, but not necessarily in the binding of the substrate, whereas the dramatically different inhibition constants for Arg-hydroxamate binding to the metal-substituted derivatives indicates direct binding of this inhibitor to the metal center. The activity-pH profiles of serralysin and its Co2+ and Cu2+ derivatives and the optical-pH profile of Cu-serralysin have been obtained, in which the decrease in activity at higher pH values was found to be associated with a dramatic increase in the Tyr-to-Cu2+ charge transfer transitions. This observation suggests that the binding of Tyr216 to the metal is inhibitory. A metal-centered mechanism is proposed for serralysin catalysis based on the results presented here, in which the detachment of the coordinated Tyr and formation of a H-bond with the transition-state complex are considered essential for the stabilization of the transition state.

Streptomyces dizinc aminopeptidase (sAP) shows a specific activity of 33.7 nmol min(-1) mg(-1) toward the hydrolysis of the transition-state analogue bis-p-nitrophenylphosphate with a rate constant of k(cat)/K(m)=100 M(-1) s(-1) and a first-order rate enhancement of about 10(10), which is much superior to several Zn chemical models and comparable to some phosphodiesterases. This study suggests that sAP can serve as a novel dinuclear model system to provide further insight into dinuclear hydrolysis.

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