Direct Brønsted Analysis of the Restoration of Activity to a Mutant Enzyme by Exogenous Amines

Abstract

A true Brønsted analysis of proton transfer in an enzyme mechanism is made possible by the chemical rescue of an inactive mutant of aspartate aminotransferase, where the endogenous general base, Lys258, is replaced with Ala by site-directed mutagenesis. Catalytic activity is restored to this inactive mutant by exogenous amines. The eleven amines studied generate a Brønsted correlation with beta of 0.4 for the transamination of cysteine sulfinate, when steric effects are included in the regression analysis. Localized mutagenesis thus allows the classical Brønsted analysis of transition-state structure to be applied to enzyme-catalyzed reactions.

Aminolevulinate synthase: Lysine 313 is not essential for binding the pyridoxal phosphate cofactor but is essential for catalysis

“…One possible candidate for the catalytic base is the e-NH2 moiety of Lys-313. This situation, where the active-site Lys has a catalytic role, has been observed with other PLP-dependent enzymes (Toney & Kirsch, 1989;Nishi-mura et al, 1991;Lu et al, 1993). For example, the €-amino group of Lys-258 of L-aspartate aminotransferase (i.e., the active-site Lys) catalyzes the proton transfer in the tautomerization of the external aldimine to ketimine (Kirsch et al, 1984;Toney & Kirsch, 1989).…”

“…They have been shown to increase the reactivity of the P L P 4"aldehyde group, facilitating the formation of an external aldimine between the substrate and the PLP cofactor (Cordes & Jencks, 1962). In addition, lysine residues have also been shown to be involved in catalysis (Toney & Kirsch, 1989;Lu et al, 1993), in formation of enzymesubstrate intermediates and product release (Lu et al, 1993), and in cofactor binding (Nishimura et al, 1991;Lu et al, 1993).…”

“…Lysine residues involved in internal aldimines in PLPdependent enzymes have been proposed to have a multitude of roles (Cordes & Jencks, 1962;Toney & Kirsch, 1989;Nishimura et al, 1991;Lu et al, 1993). They have been shown to increase the reactivity of the P L P 4"aldehyde group, facilitating the formation of an external aldimine between the substrate and the PLP cofactor (Cordes & Jencks, 1962).…”

Structure of the transition state for the binding of c-Myb and KIX highlights an unexpected order for a disordered system

“…The slope of the observed correlation, classically denoted as α, reflects the position of the transition state along the reaction coordinate. Surprisingly, LFER plots not only apply to simple organic reactions but also to complex reactions stabilized by many noncovalent interactions, and have been then used in enzymology (25), binding reactions involving allosteric control (26,27), and protein folding (28).…”

The Different Inhibition Mechanisms of OXA-1 and OXA-24 β-Lactamases Are Determined by the Stability of Active Site Carboxylated Lysine

“…Thus, in BlaR1 sensor, if -nitrogen protonation can be prevented, Lys-62 will remain carboxylated, and the acyl-enzyme can be hydrolyzed. Based on this consideration, Birck et al (47) changed the Lys-62 in BlaR1 to S-(4-butanoate)-cysteine by chemical mutagenesis (53) to see whether it is sufficient to convert the BlaR1 sensor from a susceptible receptor to an antibiotic-resistant enzyme (51). The results show that the cysteine derivative does not undergo decarboxylation, and the variant hydrolyzes the acyl-enzymes formed from a broad spectrum of antibiotics (51).…”