Analysis of the molecular relatedness of four extended spectrum β-lactamases (SHV-2, SHV-3, SHV-4 and SHV-5) by comparative protein titration curves

Abstract

Six β-lactamases from Klebsiella pneumoniae, five of which (SHV-1, SHV-2, SHV-3, SHV-4 and SHV-5) were plasmid-encoded and one which (βla GN 11–03) was chromosomally-encoded, were compared by analysis of their isoelectric points (pI), electrophoretic mobilities (M_F) and titration curves or pH gradient electrophoresis. Four groups were denned by their pI and M_F, namely SHV-1 and SHV-2 (pI = 7·6, M_F ⌣ 14), SHV-3 and 01a GN 11-03 (pi - 7·0, M_F ⌣ 20), SHV-4 (pI = 7·8 M_F ⌣ 12) and SHV-5 (pI = 8·2, M_F ⌣ 5). The titration curves of SHV-1 and SHV-2 enzymes on the one hand, and SHV-3 and β1a GN 11–03 on the other were completely superimposable for the whole of the pH gradient (3·5–10), indicating strong similarity. Conservative amino-acid substitutions could account for the differences in the substrate spectra of the purified enzymes. The differences observed between the titration curves of the enzymes SHV-l/SHV-3, SHV-l/β1a GN 11–03, SHV-2/SHV-3 and SHV-5/SHV-4 pairs were consistent with the replacement of a basic amino-acid residue in the former enzyme of each pair by an acidic residue in the latter. Similarly, the titration curves of SHV-1/SHV-4 and of SHV-2/SHV-4 pairs may suggest the replacement of an acidic amino-acid in the former β-lactamases by a neutral amino-acid in the latter of each pair. However, the presence of several self-cancelling or neutral substitutions is also possible. In contrast, when SHV-1 and TEM-1 (pI = 5·4 M_F ⌣ 45) were titrated together, no structural relationship could be inferred.