CATALYTIC AND CONFORMATIONAL PROPERTIES OF CROSS-LINKED DERIVATIVES OF PENICILLINASE

Exopenicillinase (penicillin amido-β-lactamhydrolase, EC 3.5.2.6) has been isolated from the culture supernatant of Bacillus cereus, strain 569/H. Crosslinking of the enzyme by covalent attachment of bifunctional reagents to non-essential amino acid residues yielded catalytically active derivatives with altered properties. The reagents used include cyanuric chloride, hexamethylene diisocyanate, diethyl malonimidate and glutaraldehyde at several concentrations. All derivaties showed a marked increased in thermostability consistent with a general stabilization of the native conformation of the enzyme. Further consequences of cross-linking were studied in some detail in the glutaraldehyde derivatives. The native enzyme responds to the presence of certain substrates (A-type penicillins) by a characteristic change in conformation and in reaction kinetics. The effects of such substrates can be virtually eliminated by extensive cross-linking. Similarly, the antigenic identity of the native enzyme appeared to be lost in the glutaraldehyde derivative, although it was not altered by an analogous modification with a monofunctional reagent. Surprisingly, the Michaelis constants of the enzyme were not affected by cross-linking. The results are discussed in terms of the effect of constraint of conformational flexibility on the behavior of the enzyme. © 1979.