Abstract
The SHV‐type β‐lactamase SHV‐2A is related to SHV‐1 by a Gly‐238‐Ser replacement. Strains carrying SHV‐2A are resistant to the third generation cephems cefotaxime and ceftizoxime, whereas those that carry SHV‐1 are sensitive to these drugs. We present a kinetic analysis of a SHV‐1 and SHV‐2A enzymes, with the goal of gaining insight into the role of residue 238 in hydrolyzing cefotaxime and ceftizoxime. SHV‐2A shows altered kinetic properties for a number of other cephems that also have heterocyclic side chains at the amino position of the 7‐aminocephalosporanic acid nucleus (R~1~ side chain), including a significantly higher k~cat~/K~m~ than does SHV‐1 for cephaloridine, cephalothin, and cefotiam. Two cephems with straight chain R~1~ substitutions, cephalosporin C and cephacetrile, are not hydrolyzed more efficiently by SHV‐2A. These results indicate that the Ser‐238‐Gly substitution increases the affinity toward cephems with a heterocyclic ring in the R~1~ side chain. In addition, the data for ampicillin and benzylpenicillin show that addition of a nitrogen to the second carbon of the R~1~ side chain of a penem results in a lower k~cat~/K~m~ for SHV‐2A relative to SHV‐1. These data strongly suggest that the previously proposed hydrogen bond formation between Ser‐238 and the second carbon nitrogen of cefotaxime is not an important factor in hydrolysis by SHV‐2A. We propose that the Gly‐238 to Ser‐238 replacement in SHV‐2A has altered the hydrophobic pocket so that it can better accommodate cephems with bulky R~1~ side chains.