Protein Engineering, Vol. 13, No. 4, 267-274,
April 2000
© 2000 Oxford University Press
Structure–function analysis of 
-helix H4 using PSE-4 as a model enzyme representative of class A ß-lactamases
1 Microbiologie Moléculaire et Génie des Protéines, Pavillon Charles-Eugène Marchand, Département de Biologie Médicale, Faculté de Médecine, Université Laval, Ste-Foy, Québec, Canada, G1K 7P4, 2 Department of Biochemistry, Baylor College of Medicine, Houston, TX 77030, USA and 3 Département de Chimie, Pavillon Vachon, Faculté des Sciences et de Génie, Université Laval, Ste-Foy, Québec, Canada, G1K 7P4
We extracted maximum information for structure–function analysis of the PSE-4 class A ß-lactamase by random replacement mutagenesis of three contiguous codons in the H4 
-helix at amino acid positions Ala125, Thr126, Met127, Thr128 and Thr129. These positions were predicted to interact with suicide mechanism-based inhibitors when examining the PSE-4 three-dimensional model. Structure–function studies on positions 125–129 indicated that in PSE-4 these amino acids have a role distinct from those in TEM-1, in tolerating substitutions at Ala125 and being invariant at Met127. The importance of Met127 was suspected to be implicated in a structural role in maintaining the integrity of the H4 -helix structure together, thus maintaining the important Ser130–Asp131–Asn132 motif positioned towards the active site. At the structural level, the H4 region was analyzed using energy minimization of the H4 regions of the PSE-4 YAM mutant and compared with wild-type PSE-4. The Tyr 125 of the mutant YAM formed an edge to face 
– interaction with Phe 124 which also interacts with the Trp 210 with the same interactions. Antibiotic susceptibilities showed that amino acid changes in the the H4 -helix region of PSE-4 are particularly sensitive to mechanism based-inhibitors. However, kinetic analysis of PSE-4 showed that the two suicide inhibitors belonging to the penicillanic acid sulfone class, sulbactam and tazobactam, were less affected by changes in the H4 -helix region than clavulanic acid, an inhibitor of the oxypenam class. The analysis of H4 -helix in PSE-4 suggests its importance in interactions with the three clinically useful inhibitors and in general to all class A enzymes.
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