ABSTRACT The catalytic activity of β-lactamases, particularly class A enzymes like KPC-2, is central to β-lactam antibiotic resistance. While phosphate buffers are widely used in enzymatic assays due to their physiological relevance, their potential to interfere with enzyme function remains underappreciated. Here, we demonstrate that phosphate acts as a competitive inhibitor of KPC-2 β-lactamase, significantly reducing catalytic efficiency in a concentration-dependent manner. This inhibition is mediated through interactions with threonine 237, as substitution with glycine (T237G) abolishes the inhibitory effect. In contrast, CTX-M-14 and TEM-1, which possess serine and alanine at the same position, respectively, exhibit minimal phosphate sensitivity, underscoring enzyme-specific buffer effects. Structural and kinetic analyses indicate that phosphate binding at the active site primarily impairs substrate affinity and, in some cases, also reduces catalytic turnover. These findings highlight the potential impact of buffer selection in β-lactamase assays and suggest that phosphate-mediated inhibition may lead to underestimation of enzyme activity and inhibitor potency, particularly in studies involving KPC-2. Standardizing assay conditions is essential for accurate evaluation of β-lactamase function and resistance mechanisms.
Ngango et al. (Fri,) studied this question.