A comparison of Zn(II) and Co(II) in the kinetics of inactivation of aminoacylase by 1,10-phenanthroline and reconstitution of the apoenzyme

Abstract

The kinetics of reconstitution of apoacylase with either Zn(II) or Co(II) and the inactivation of the Co(II) reconstituted enzyme by 1,10-phenanthroline (OP) has been studied by following the substrate reaction continuously in presence of the metal ion or OP respectively. Although the native Zn(II)-containing and the Co(II)-reconstituted enzymes have closely similar Michaelis constants and maximal velocities, the kinetics for both the inactivation by OP and the reconstitution of the apoenzyme with the metal ions differs considerably. For Co(II), both the inactivation by OP and the reconstitution show simple kinetics, but for Zn(II), the inhibition by OP is a multi-phasic process [Wang, Wu, Wang, Zhou and Tsou (1992) Biochem. J. 281, 285-290], and the kinetics of reconstitution is also much more complicated. Both the native and the Co(II)-reconstituted enzymes are inhibited by excess of Zn(II), but not by Co(II). The inhibition by Zn(II) in excess and the reconstitution of the apoenzyme with Zn(II) are co-operative processes. The inhibition by Zn and its effect on the fluorescence emission of 1-anilinonaphthalene-8-sulphonic acid bound to the native enzyme indicate multiple Zn(II)-binding sites.

Characterization of an Aminoacylase from the Hyperthermophilic Archaeon Pyrococcus furiosus

“…This is typically zinc, but cobalt, manganese, and nickel are also usually effective. The exact role of the metal is not clear, although in some of these enzymes, it is thought to have a structural role, as well as a catalytic one (59). Unfortunately, biochemical analyses of this group of enzymes have been limited mainly to kinetic studies (11,55,59,60) and detailed structural information, such as that obtained by crystallography, is not yet available (46,54).…”

“…The exact role of the metal is not clear, although in some of these enzymes, it is thought to have a structural role, as well as a catalytic one (59). Unfortunately, biochemical analyses of this group of enzymes have been limited mainly to kinetic studies (11,55,59,60) and detailed structural information, such as that obtained by crystallography, is not yet available (46,54). Because of their biotechnological potential, there have been many attempts to stabilize various types of aminoacylases, either by immobilization or by obtaining the enzyme from thermophilic sources (5,6,7).…”

Inhibition of aminoacylase 3 protects rat brain cortex neuronal cells from the toxicity of 4-hydroxy-2-nonenal mercapturate and 4-hydroxy-2-nonenal

“…Since Co 2+ does not activate AA1 (Wu, Tsou, 1993), AA1 activity was assayed without this metal ion. The enzyme was homogeneous under SDS-PAGE and had the K cat value with a typical AA1 substrate N-acetyl-L-methionine of ~20 s −1 , which is similar to the reported values for porcine and human AA1 (Lindner et al, 2008).…”

“…An important structure/functional property of AA1 and AA3 is that they both are metalloenzymes inactivated by chelating agents (Anders, Dekant, 1994; Kördel, Schneider, 1977; Lindner et al, 2003, 2008; Perrier et al, 2005; Tsirulnikov et al, 2009; Wu, Tsou, 1993). Human AA1 expressed in E. coli contained 2 zinc atoms in the active site (Lindner et al, 2003) although Co 2+ and Mn 2+ substituted for zinc in AA1 without a substantial loss of activity.…”

Kinetics of the course of reactivation of aminoacylase reconstituted using Mn2+ ions

“…The reactivation of apoenzyme reconstituted using metal ions has been described by Wu and Tsou (1993) by the following scheme:…”