The family of bacterial enzyme aminoglycoside 3'-phosphotransferases [(APH-(3')s] confers resistance to aminoglycoside antibiotics by phosphorylating these antibacterial agents. Such resistance is readily acquired by susceptible bacterial strains from resistant ones via exchange of plasmids encoding APH(3') enzymes. Aminoglycoside resistance and its spread pose serious obstacles to antibacterial therapy by these agents. The APH-(3')-type II from Gram-negative bacteria has been purified to homogeneity in milligram quantity in the laboratory of the Principal Investigator. A systematic three-pronged approach to the studies of the mechanism and structure of this APH(3') has been outlined. (i) The enzyme will be characterized in its catalytic turnover of three substrates; the kinetic parameters will be measured and the effect of pH on these parameters will be investigated. In the process, it will be determined whether APH(3')-II follows a random-order, compulsory-order or ping-pong mechanism pathway. (ii) The structure of the enzyme, especially at the active site , will be probed. A series of experiments are proposed to identify the presence and sites of disulfide linkage(s) in the protein. Furthermore, active site residues will be identified through the use of a series of four affinity inactivators based on the structure of neamine, a substrate for APH (3') enzymes. The structure of neamine would be used as a """"""""molecular template"""""""" in the preparation of these inactivators, each of which is expected to allow for the mapping of a different subsite in the APH(3') active site, after protein inactivation. In addition, a nitro analogue of neamine has been proposed as the first potential mechanism-based inactivator for this class of enzymes. This molecule is hoped to be a dual action compound that not only inactivates the enzyme, but may also retain its antibacterial activity. A detailed approach to kinetic characterization of these inactivators is outlined. We have described a novel metal-mediated protein fragmentation chemistry for identification of residues within the metal-binding site of carboxypeptidase A. This strategy will be applied to the fragmentation of APH(31)-II in our studies of the Mg2+, binding site. (iii) In collaboration with the laboratory of Professor Stephen Lerner of Wayne State University School of Medicine, point mutants of APH(31)-II will be generated at sites that would be identified. We hope to delineate the roles of the identified amino acids in binding and catalysis through kinetic studies of the mutant enzymes. The kinetics of a point mutant (Glu-182-Asp), reported recently in the literature, will be studied in the immediate future.
