Substrate specificities and structure-activity relationships for the nucleotidylation of antibiotics catalyzed by aminoglycoside nucleotidyltransferase 2''-I.
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 kinetics 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., & 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 net 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 greater than gentamicins greater than 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Gates
C A
CA
Division of Pharmaceutical Biochemistry, School of Pharmacy, University of Wisconsin, Madison 53706.
Northrop
D B
DB
D000617:Aminoglycosides; D000900:Anti-Bacterial Agents; D003854:Deoxyribonucleotides / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D008958:Models, Molecular; D008968:Molecular Conformation; D009713:Nucleotidyltransferases / Q000378:metabolism*; D012265:Ribonucleotides / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D000617:Aminoglycosides; D000900:Anti-Bacterial Agents; D003854:Deoxyribonucleotides; D012265:Ribonucleotides; D009713:Nucleotidyltransferases; C020565:gentamicin 2''-nucleotidyltransferase