Substrate specificities and structure-activity relationships for the nucleotidylation of antibiotics catalyzed by aminoglycoside nucleotidyltransferase 2"-I

Publisher Website
Google Scholar
Abstract
Aminoglycoside nucleotidyltransferase 2"-I (formerly gentamicin adenylyltransferase) conveys antibiotic resistance to Gram-negative bacteria by transfer of AMP to the 2"-hydroxyl group of 4,6-substituted deoxystreptamine-containing aminoglycosides. The kinetic constants of thirteen aminoglycoside antibiotics and the magnesium chelates of eight nucleotide triphosphates were determined with purified enzyme. Eleven of the antibiotics exhibit substrate inhibition attributed to secondary binding of the aminoglycoside to an enzyme-AMP-aminoglycoside complex. Maximal velocities vary by only 4-fold, versus variation of values of Vmax/Km for the aminoglycosides of nearly 4000-fold, consistent with a Theorell-Chance kinetic mechanism as proposed for this enzyme [Gates, C. A., and Northrop, D. B. (1988) Biochemistry (second of three papers in this issue)] with the added specification that the binding of aminoglycosides is in rapid equilibrium. Under these conditions, Vmax/Km becomes kcat/Kd, where kcat is the rate constant for catalysis (but not turnover) and Kd is the dissociation constant of aminoglycosides from a complex with enzyme and nucleotide. Values of kcat fall closely together into three distinct sets, with the 3'',4''-dideoxygentamicins > gentamicins > kanamycins. These sets reflect unusual structure-activity correlations which are specific for catalysis but have nothing to do with the maximal velocity of this enzyme. The contribution of individual functional groups to binding was evaluated according to Kd values generated from substrate inhibition; specifically, bvinding is reduced by esterification at the 6"-carbon, hydroxylation of the 2''-carbon, unsaturation of the 4'',5''- carbon-carbon bond, methylation of the 6''-carbon or the 6''-amino groups, and ethylation of the 1-amino group. Comparisons between gentamicins and kanamycins are inconsistent with a common site of adenylylation at the 2"-hydroxyl but suggest either the 3''- or 4''-hydroxyl of the former. Unfortunately for the search for better antibiotics, most structure-activity relationships of enzymatic activity parallel antibiotic activity, with two exceptions being alkylation of the 1-amino group and stereochemical repositioning of the 5-hydroxyl group.

This publication has 10 references indexed in Scilit: