Substitutions in the Ω‐Loop of TEM β‐Lactamase Change the Substrate Profile of the Enzyme (LB140)

Serine β‐lactamases are bacterial enzymes that hydrolyze β‐lactam antibiotics. Mechanistically, serine β‐lactamases are similar to serine proteases such as chymotrypsin. They both utilize acylation and deacylation of an active site serine in their mechanism of catalysis. TEM‐1, a common plasmid‐encoded serine β‐lactamase, catalyzes the hydrolysis of early penicillins and cephalosporins. Here we examine a previously identified triple mutant of TEM‐1, 165‐TyrTyrGly‐167 (wild type165‐TrpGluPro‐167) with substrate specificity altered from preferential hydrolysis of ampicillin to that of ceftazidime. When compared to chymotrypsin, the Glu166Tyr substitution in the TEM‐1 triple mutant is analogous to a substitution of the general base His57 in chymotrypsin that results in an enzyme that maintains function. Our findings agree with previous observations of altered substrate specificity of the triple mutant, which displays increased hydrolysis of ceftazidime. Additionally, enzyme kinetic analyses show that the hydrolysis of ceftazidime follows a branched pathway characteristic of substrate‐induced reversible inactivation. Structural analysis of the triple mutant reveals enlargement of the active site making it more accessible for larger substrates. Furthermore, in the crystal structure of the triple mutant, Tyr166 is within hydrogen bond distance to Ser70 suggesting involvement in the catalytic mechanism of the enzyme. These findings indicate that Glu166 general base residue can be substituted in the mechanism of serine β‐lactamases. More generally, this provides insights into alternate mechanisms for reactions catalyzed by serine hydrolases. Grant Funding Source : Supported by NIH