Carbapenems and SHV-1 β-Lactamase Form Different Acyl-Enzyme Populations in Crystals and Solution

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Abstract
The reactions between single crystals of the SHV-1 β-lactamase enzyme and the carbapenems, meropenem, imipenem, and ertapenem, have been studied by Raman microscopy. Aided by quantum mechanical calculations, major populations of two acyl-enzyme species, a labile Δ2-pyrroline and a more tightly bound Δ1-pyrroline, have been identified for all three compounds. These isomers differ only in the position of the double bond about the carbapenem nucleus. This discovery is consonant with X-ray crystallographic findings that also identified two populations for meropenem bound in SHV-1: one with the acyl C═O group in the oxyanion hole and the second with the acyl group rotated 180° compared to its expected position [Nukaga, M., Bethel, C. R., Thomson, J. M., Hujer, A. M., Distler, A. M., Anderson, V. E., Knox, J. R., and Bonomo, R. A. (2008) J. Am. Chem. Soc. (in press)]. When crystals of the Δ1- and Δ2-containing acyl-enzymes were exposed to solutions with no carbapenem, rapid deacylation of the Δ2 species was observed by kinetic Raman experiments. However, no change in the Δ1 population was observed over 1 h, the effective lifetime of the crystal. These observations lead to the hypothesis that the stable Δ1 species is due to the form seen by X-ray with the acyl carbonyl outside the oxyanion hole, while the Δ2 species corresponds to the form with the carbonyl inside the oxyanion hole. Soak-in and soak-out Raman experiments also demonstrated that tautomeric exchange between the Δ1 and Δ2 forms does not occur on the crystalline enzyme. When meropenem or ertapenem was reacted with SHV-1 in solution, the Raman difference spectra demonstrated that only a major population corresponding to the Δ1 acyl-enzyme could be detected. The 1003 cm−1 mode of the phenyl ring positioned on the C3 side chain of ertapenem acts as an effective internal Raman intensity standard, and the ratio of its intensity to that of the 1600 cm−1 feature of Δ1 provides an estimate of the relative populations of Δ1. In solution, I1600/I1003 equals 2, and in the crystal, I1600/I1003 equals 1. This is strong evidence that the Δ1 and Δ2 acyl-enzymes in the crystal are present in approximately equal amounts, in agreement with the X-ray data. However, in solution there are twice as many Δ1 species per Phe group, and this represents approximately 100% of the active sites, which is consistent with the observed inhibition of the enzyme’s activity.

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