Project Summary/Abstract b-lactam antibiotics have a broad spectrum of anti-bacterial activity including important Gram-positive and negative pathogens. b-lactams are the most widely used antibiotics and include penicillins, cephalosporins and carbapenems. Bacterial resistance to these drugs, however, has been steadily increasing, significantly reducing treatment options. The most common mechanism of resistance is b-lactamase-catalyzed hydrolysis, which renders the antibiotics ineffective. Carbapenems are used as antibiotics of last resort. The increasing use of these drugs, however, has led to the emergence of carbapenemase enzymes. This group includes the KPC-2 b-lactamase that effectively hydrolyzes nearly all b-lactam antibiotics and has spread globally to multiple species of Enterobacteriaciae. K. pneumoniae strains producing KPC are a particular concern, as the mortality rate for infections from such strains is 30-40%. The design of new antibiotics that escape hydrolysis by KPC enzymes is a challenge. To achieve this, it is necessary to understand the catalytic mechanism and basis for substrate specificity of the enzyme and how this relates to inhibitor design. This, in turn, requires an understanding of the affinity of binding of the antibiotic/inhibitor as well as the rates of the chemical steps. Further, it is necessary to understand how the structure and dynamics of the enzyme influence drug binding and hydrolysis. Finally, for future antibiotic design, it is important to understand how changes in antibiotic structure impact binding affinity and the rates of chemical steps. We propose to achieve these goals with KPC-2 and variants of the enzyme that have an extended substrate profile or provide resistance to the antibiotic/inhibitor combination ceftazidime/avibactam. While there has been extensive characterization of b-lactamase structure and steady-state enzyme kinetics, there is a large gap in understanding the rates of individual steps of b-lactamase-catalyzed hydrolysis of b-lactam antibiotics. We will address this gap by determining the chemical and structural basis of KPC-2 catalysis with a combination of pre-steady state kinetic, X-ray crystallographic, and molecular dynamics simulation approaches. Aim 1 will provide a mechanistic and structural basis for the hydrolysis of b-lactam antibiotics by the KPC-2 enzyme. The studies in Aim 2 will characterize the kinetic and structural basis of increased hydrolysis of the extended-spectrum cephalosporin, ceftazidime, by drug-resistant KPC variants. Finally, Aim 3 will determine the structural and mechanistic basis of action of KPC-variant enzymes that are resistant to the ceftazidime/avibactam inhibitor combination. Our multidisciplinary approach is expected to answer these questions and lead to new concepts and tools to address the problem of antibiotic/inhibitor resistance.