A “Moonlighting” Di-Zinc Aminopeptidase from Streptomyces griseus: 

Mechanisms for Peptide Hydrolysis and the 40-Billion-Fold Acceleration of the Alternative Phosphodiester Hydrolysis by This Enzyme


Altan Ercan,# Hyun Ik Park,^ and Li-June Ming*

Department of Chemistry and Institute for Biomolecular Science

University of South Florida

4202 E. Fowler Avenue, CHE205

Tampa, Florida 33620-5250, USA


  This work was supported by the Petroleum Research Funds administrated by the American Chemical Society (ACS-PRF #35313-AC3) and the National Institute of Health (GM064400-01A2). 


# Current address: Department of Infectology, The Scripps Institute, 5353 Parkside Dr.,

Jupiter, FL 33458, USA

^ Current address: R & D Systems, Inc., 614 McKinley Place N.E., Minneapolis, MN 55413



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]. Since tetrahedral phospho-centers often serve as transition-state inhibitors toward the hydrolysis of 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, 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 detailed investigation of both peptide and phosphodiester hydrolyses catalyzed by SgAP.  The latter exhibits a first-order rate enhancement of 40 billion-fold compared to the uncatalyzed reaction at pH 7.0 and 25 °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.  Better understanding of enzyme catalytic promiscuity is also expected to shed light on evolution and action of enzymes.