Clavulanic acid is a widely used β-lactamase inhibitor whose key β-lactam core is formed by β-lactam synthetase. β-Lactam synthetase exhibits a Bi-Ter mechanism consisting of two chemical steps, acyl-adenylation followed by β-lactam formation. 32;PP i-ATP exchange assays showed the first irreversible step of catalysis is acyl-adenylation. From a small, normal solvent isotope effect (1.38 ± 0.04), it was concluded that β-lactam synthesis contributes at least partially to k cat. Site-specific mutation of Lys-443 identified this residue as the ionizable group at pK a ∼ 8.1 apparent in the pH-k cat profile that stabilizes the β-lactam-forming step. Viscosity studies demonstrated that a protein conformational change was also partially rate-limiting on k cat attenuating the observed solvent isotope effect on β -lactam formation. Adherence to Kramers' theory gave a slope of 1.66 ± 0.08 from a plot of log(°k cat/k cat) versus log(η/η°) consistent with opening of a structured loop visible in x-ray data preceding product release. Internal "friction" within the enzyme contributes to a slope of > 1 in this analysis. Correspondingly, earlier in the catalytic cycle ordering of a mobile active site loop upon substrate binding was manifested by an inverse solvent isotope effect (0.67 ± 0.15) on k cat/K m. The increased second-order rate constant in heavy water was expected from ordering of this loop over the active site imposing torsional strain. Finally, an Eyring plot displayed a large enthalpic change accompanying loop movement (ΔH∼ 20 kcal/mol) comparable to the chemical barrier of β -lactam formation.