Class C β-Lactamases Operate at the Diffusion Limit for Turnover of Their Preferred Cephalosporin Substrates

Abstract

It has been suggested that class C β-lactamases have evolved to carry out a metabolic reaction other than hydrolysis of β-lactam antibiotics. It is demonstrated in the present study that the class C β-lactamase from Enterobacter cloacae P99 has reached the diffusion limit in its ability to hydrolyze its preferred cephalosporin substrates. The increase in the solution viscosity by addition of a microviscogen (sucrose) caused the decline in the parameterk cat/Km for hydrolysis of cephaloridine and cephalosporin C (approximately 2.5-fold at a relative viscosity of 2.9). A similar increase in viscosity has no effect on the turnover rate of the poorer substrates cefepime and penicillin G. Addition of a macroviscogen (polyethylene glycol) to the reaction mixture did not change the rate of turnover for any of the substrates tested because in this case the viscogen would not interfere with the motion of small molecules, as was expected. Therefore, it would appear that the driving force behind the evolution of this class C β-lactamase and, in principle, other enzymes of this class is indeed the functional reaction of this enzyme as a drug resistance factor.

Enzyme efficiency but not thermostability drives cefotaxime resistance evolution in TEM-1 β-lactamase

“…Another possible explanation for the observed saturation is that some of our best β-lactamase alleles may approach the diffusion limit, as previously observed for TEM-1 in other bacterial species (Hardy and Kirsch 1984; Christensen et al 1990; Bulychev and Mobashery 1999). Classically (Albery and Knowles 1976), this condition requires k cat / K M values on the order of 10 8 –10 9 M − 1 s − 1 .…”

“…However, it has also long been appreciated that viscosity resulting from macromolecular crowding within bacterial cells can reduce this threshold by two log-orders (Benner 1989). Moreover, enzymatic velocity is attenuated in a continuous fashion as k cat / K M approaches the relevant physical diffusion-limitation rate (Brouwer and Kirsch 1982; Hardy and Kirsch 1984; Bulychev and Mobashery 1999). As the mean k cat / K M among 238S alleles is 2.78 × 10 5 M − 1 s − 1 (though only 1.11 × 10 3 M − 1 s − 1 for G238 alleles; table 1), it seems reasonable to suppose that in vivo diffusion may further contribute to the fact that for large values of k cat / K M , observed MIC values are lower than predicted.…”

Effect of Sugar Molecules on the Viscosity of High Concentration Monoclonal Antibody Solutions

“…Although sugars have been used as microviscogen agents to mediate enzymatic reaction rates by increasing solution viscosity (16,17), to the best of our knowledge, the effect of sugars on solution viscosity at high protein concentrations has not been fully characterized. These types of studies are complicated by the fact that the traditional rheological techniques, such as the cone-and-plate method, require significant amounts of material and time (3,(18)(19)(20).…”

Effects of the Val211Gly substitution on molecular dynamics of the CMY‐2 cephalosporinase: Implications on hydrolysis of expanded‐spectrum cephalosporins

“…Hydrolysis of β‐lactams by AmpCs proceeds by a double displacement catalytic mechanism that includes a nucleophilic attack of the activated oxygen of Ser‐64 at the carbon of the β‐lactam's amide bond, the formation of acylation tetrahedral high energy species that transforms to an acyl‐enzyme complex, and the collapse of the latter complex through reaction with an activated water molecule 7. AmpCs have reached “catalytic perfection” against older cephalosporins (e.g., cephalothin) operating at the diffusion limit 8. However, their efficiency against expanded‐spectrum cephalosporins (ESCs), such as ceftazidime and cefotaxime possessing a bulky oxyimino substitution (R1 side chain) at the C7 carbon [Fig.…”