Carbapenems and SHV-1 beta-lactamase form different acyl-enzyme populations in crystals and solution

Abstract

The reactions between single crystals of the SHV-1 beta-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 Delta (2)-pyrroline and a more tightly bound Delta (1)-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 CO group in the oxyanion hole and the second with the acyl group rotated 180 degrees 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 Delta (1)- and Delta (2)-containing acyl-enzymes were exposed to solutions with no carbapenem, rapid deacylation of the Delta (2) species was observed by kinetic Raman experiments. However, no change in the Delta (1) population was observed over 1 h, the effective lifetime of the crystal. These observations lead to the hypothesis that the stable Delta (1) species is due to the form seen by X-ray with the acyl carbonyl outside the oxyanion hole, while the Delta (2) 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 Delta (1) and Delta (2) 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 Delta (1) 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 Delta (1) provides an estimate of the relative populations of Delta (1). In solution, I 1600/ I 1003 equals 2, and in the crystal, I 1600 /I 1003 equals 1. This is strong evidence that the Delta (1) and Delta (2) 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 Delta (1) 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.

Figure 1

The carbapenems meropenem, ertapenem, imipenem, and doripenem are structurally similar to the penicillins, but the sulfur atom in position 4 is replaced by a carbon atom.

Figure 2

The proposed reaction scheme between a generalized carbapenem and SHV-1, a class A β-lactamase. Acylation of an active-site residue, Ser70, results in partitioning of the carbapenem into either the Δ¹ or Δ²-pyrroline. Unfavorable positioning of the Δ¹ acyl-enzyme in the SHV-1 active site results in a hydrolytically inert species. *Proposed by Zafaralla et al. but there is no evidence of a reversible step from our crystal data(9). **This mechanism is a proposal based on the present data.

Figure 3a and 3b

(a) Raman spectra of the unbound ligands meropenem (top), imipenem, and ertapenem (bottom). (b) Raman difference spectra of the meropenem (top), imipenem, and ertapenem (bottom) bound to SHV-1. The intense modes between 1550 and 1600 cm^-1 provide detailed information arrangement of π-electrons in the carbapenem nucleus.

Figure 3a and 3b

(a) Raman spectra of the unbound ligands meropenem (top), imipenem, and ertapenem (bottom). (b) Raman difference spectra of the meropenem (top), imipenem, and ertapenem (bottom) bound to SHV-1. The intense modes between 1550 and 1600 cm^-1 provide detailed information arrangement of π-electrons in the carbapenem nucleus.

Figure 4

Ab initio calculations for molecule 1 (inset) demonstrate that the C2=C3 stretching frequency is sensitive to the C2=C3-S-C dihedral angle. While only the free ligand of imipenem exhibits rotational isomerism, complexation with the β-lactamase causes all of the carbapenems to adopt closely related rotational isomers for the Δ² acyl-enzyme in crystallo.

Figure 5

Structure of molecule 2, Δ¹-acyl enzyme mimic, and molecule 3, Δ²-acyl enzyme mimic, with predicted stretching frequencies for the C=N and C=C stretches.

Figure 6

Predicted Raman spectra for molecules 2 and 3 (Figure 5), which mimic the Δ¹ or Δ²-acyl enzyme, respectively. The dominant features arise from the highly polarizable N1=C2 stretch around 1600 cm^-1 (solid line) or C2=C3 stretching motions around 1550 cm-1 (dashed line). Hydrolysis of meropenem shows that the Δ¹-pyrroline is strongly favored thermodynamically over the Δ² isomer under basic conditions, while approximately equal amounts of the Δ¹ and Δ² isomers are present under acidic conditions.

Figure 7

Raman difference spectra of a “soak-out” experiment for meropenem (top), imipenem, and ertapenem (bottom). The dominant band at 1600 cm^-1 indicates that the Δ¹ tautomer does not deacylate from the enzyme. Features associated with protein modes, such as 1670 and 1324 cm^-1, indicate a “tightening” of the β-sheet motifs upon complexation.

Figure 8

Solution Raman difference spectra of meropenem (top) and ertapenem (bottom) bound to SHV-1 in 1:1 complex at 750 μM in 2 mM HEPES at pH 7.0.

Figure 9. Partial Raman difference spectra for ertapenem in crystal and solution

The 1003 cm^-1 mode of the phenyl ring positioned on the C3 side chain of ertapenem acts as an effective internal intensity standard and the ratio of its intensity (I) 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, whereas I₁₆₀₀/I₁₀₀₃ equals 1 in the crystal.