Dissection of the Stepwise Mechanism to β-Lactam Formation and Elucidation of a Rate-determining Conformational Change in β-Lactam Synthetase

Abstract

Clavulanic acid is a widely used ␤-lactamase inhibitor whose key ␤-lactam core is formed by ␤-lactam synthetase. ␤-Lactam synthetase exhibits a Bi-Ter mechanism consisting of two chemical steps, acyl-adenylation followed by ␤-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 ␤-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 ϳ 8.1 apparent in the pH-k cat profile that stabilizes the ␤-lactamforming step. Viscosity studies demonstrated that a protein conformational change was also partially rate-limiting on k cat attenuating the observed solvent isotope effect on ␤-lactam formation. Adherence to Kramers' theory gave a slope of 1.66 ؎ 0.08 from a plot of log( o k cat /k cat ) versus log(/ o ) 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 (⌬H ‡ ϳ 20 kcal/mol) comparable to the chemical barrier of ␤-lactam formation.Resistance to antibiotics is a consequence of their widespread use in the treatment of disease and can arise with dispiriting rapidity after the introduction of a new drug. ␤-Lactam antibiotics constitute 55% of all antibiotics sold despite their prominent use for more than 50 years (1). Their longevity is because of semi-synthetic structural modifications, the discovery of new members of the family from natural sources, and the growing importance of inhibitors of the principal means of resistance, the ␤-lactamases (2). Clavulanic acid is a potent serine ␤-lactamase inhibitor isolated from Streptomyces clavuligerus (3) whose combination with broad spectrum ␤-lactam antibiotics has proven to be highly effective against resistant infections in humans (4, 5). Inhibitor-resistant ␤-lactamases are emerging (1), however, and the need for new entities clinically useful against them is growing.Clavulanic acid is one of only three ␤-lactamase inhibitors in clinical use (Scheme 1) (6). Although other clavams produced in nature may possess antibacterial and antifungal properties, clavulanic acid is the only known clavam with potent ␤-lactamase inhibitory activity owing in part to its 3R,5R stereochemistry (7). It is therefore important to understand the steps involved in this pathway to enable the production of derivatives. Substitution at C-6 of clavulanic acid derivatives, for example, led to broader spectrum inhibition toward ␤...

Inverse Solvent Isotope Effects Arising from Substrate Triggering in the Factor Inhibiting Hypoxia Inducible Factor

“…(32, 33) Furthermore, the lack of a viscosity effect indicates FIH does not undergo a viscosity-sensitive conformational change, which has been shown to obscure SIEs for some enzymes. (34, 35)…”

Examination of Mechanism of N-Acetyl-1-d-myo-inosityl-2-amino-2-deoxy-α-d-glucopyranoside Deacetylase (MshB) Reveals Unexpected Role for Dynamic Tyrosine

“…The slopes for log k cat versus log rel plots range from 0 to 1 depending on degree of coupling of active site to solvent (25). The finding that the slopes for WT MshB obtained in the presence of glycerol and sucrose are 0.72 Ϯ 0.09 and 0.81 Ϯ 0.06, respectively, suggests that there is significant coupling between the dynamics of MshB and solvent molecules.…”

“…The rel values for 0, 10, 20, 27.5, 32.5, and 35% sucrose are 1, 1.32, 1.88, 2.48, 3.06, and 3.42, respectively (24). The rel values for 0, 10, 20, 30, and 35% glycerol are 1, 1.3, 1.7, 2.3, and 2.9, respectively (25). The rel values for 0, 5, and 10% Ficoll 400 are 1, 2.2, and 4.5, respectively (26).…”

Escherichia coli Heptosyltransferase I: Investigation of Protein Dynamics of a GT-B Structural Enzyme

“…12 Specifically, the effects of microviscogens glycerol and ethylene glycol on HepI k cat (0.3 s −1 ) were compared with the effect of macroviscogen PEG 8000. The data, plotted as the log of reciprocal relative rates versus log of relative viscosity, yielded δ values of 0.93, 1.0, and 0.12 for glycerol and ethylene glycol and PEG 8000, respectively (Figure S2).…”