4r3b

Publication Abstract from PubMed

For the class A beta-lactamase SHV-1, the kinetic and mechanistic properties of the clinically used inhibitor sulbactam are compared with the sulbactam analog substituted in its 6beta position by a CH2OH group (6beta-(hydroxymethyl)penicillanic acid). The 6beta substitution improves both in vitro and microbiological inhibitory properties of sulbactam. Base hydrolysis of both compounds was studied by Raman and NMR spectroscopies and showed that lactam ring opening is followed by fragmentation of the dioxothiazolidine ring leading to formation of the iminium ion within 3 min. The iminium ion slowly loses a proton and converts to cis-enamine (which is a beta-aminoacrylate) in 1 h for sulbactam and in 4 h for 6beta-(hydroxymethyl) sulbactam. Rapid mix-rapid freeze Raman spectroscopy was used to follow the reactions between the two sulfones and SHV-1. Within 23 ms, a 10-fold excess of sulbactam was entirely hydrolyzed to give a cis-enamine product. In contrast, the 6beta-(hydroxymethyl) sulbactam formed longer-lived acyl-enzyme intermediates that are a mixture of imine and enamines. Single crystal Raman studies, soaking in and washing out unreacted substrates, revealed stable populations of imine and trans-enamine acyl enzymes. The corresponding X-ray crystallographic data are consonant with the Raman data and also reveal the role played by the 6beta-hydroxymethyl group in retarding hydrolysis of the acyl enzymes. The 6beta-hydroxymethyl group sterically hinders approach of the water molecule as well as restraining the side chain of E166 that facilitates hydrolysis.

Detecting a Quasi-stable Imine Species on the Reaction Pathway of SHV-1 beta-Lactamase and 6beta-(Hydroxymethyl)penicillanic Acid Sulfone.,Che T, Rodkey EA, Bethel CR, Shanmugam S, Ding Z, Pusztai-Carey M, Nottingham M, Chai W, Buynak JD, Bonomo RA, van den Akker F, Carey PR Biochemistry. 2015 Jan 8. PMID:25536850^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.