Mechanistic studies of the astacin-like Serratia metalloendopeptidase serralysin: highly active (>2000%) Co(II) and Cu(II) derivatives for further corroboration of a "metallotriad" mechanism

Abstract

Serralysin is a bacterial Zn-endopeptidase which has been considered a virulence factor to cause tissue damage and anaphylactic response. It contains a coordinated Tyr that is unique to the astacin-like Zn enzymes. The coordinated Tyr has been proposed to play an important role in the action of this endopeptidase family. Several metal-substituted derivatives of serralysin (including Mn2+, Co2+, Ni2+, Cu2+, and Cd2+ derivatives) are found to exhibit significant activities. Particularly, the Co- and Cu-substituted derivatives exhibit much higher activities than the native serralysin toward the hydrolysis of the tripeptide mimic benzoyl-Arg- p-nitroanilide, i.e., 35 and 49 times higher in k(cat) and 33 and 26 times in k(cat)/ K(m), respectively. Such remarkably higher activities of metal-substituted derivatives, especially the Cu derivative, than that of the native Zn enzyme are rare in the literature, reflecting the uniqueness of this enzyme among all Zn enzymes. The significantly different k(cat) yet similar K(m) values among the several metal derivatives suggests that the metal center is involved in catalysis, but not necessarily in the binding of the substrate, whereas the dramatically different inhibition constants for Arg-hydroxamate binding to the metal-substituted derivatives indicates direct binding of this inhibitor to the metal center. The activity-pH profiles of serralysin and its Co2+ and Cu2+ derivatives and the optical-pH profile of Cu-serralysin have been obtained, in which the decrease in activity at higher pH values was found to be associated with a dramatic increase in the Tyr-to-Cu2+ charge transfer transitions. This observation suggests that the binding of Tyr216 to the metal is inhibitory. A metal-centered mechanism is proposed for serralysin catalysis based on the results presented here, in which the detachment of the coordinated Tyr and formation of a H-bond with the transition-state complex are considered essential for the stabilization of the transition state.

Overexpression and Mechanistic Characterization of Blastula Protease 10, a Metalloprotease Involved in Sea Urchin Embryogenesis and Development

“…6, A and D), indicating the presence of two ionizable groups in the catalytic mechanism of Zn-BP10. Two ionization constants, pK a1 ϭ 5.94 and pK a2 ϭ 10.2, were obtained from the k cat versus pH profile, and pK a1 ϭ 6.3 and pK a2 ϭ 10.1 were obtained from the k cat /K m versus pH profile, which can be assigned to the deprotonation of a zinc-bound water and the coordinated tyrosine, respectively, which are consistent with previous reports for serralysin (24). The role of the coordinated Tyr in BP10 catalysis is further addressed below.…”

“…Thus, the Zn 2ϩ is frequently replaced with other spectroscopically active metal ions to afford derivatives that can offer detailed insight into the catalytic mechanisms and structure within the active site (36). The formation of the Cu 2ϩ -substituted BP10 (Cu-BP10) is evident by the intense Tyr-to-Cu 2ϩ LMCT at 454 nm, analogous to that in Cu 2ϩ -astacin and Cu 2ϩ -serralysin (24,37). The activity of Cu-BP10 is considerably higher than Zn-BP10 in terms of k cat (0.76 s Ϫ1 ) and k cat /K m (5430 M Ϫ1 s Ϫ1 ), reflecting a 960% increase in activity in terms of k cat and 485% in terms of k cat /K m .…”

“…However, the coordination of the Tyr-phenolate under physiological conditions does not seem to affect astacin catalysis under neutral conditions. The status of the coordinated tyrosine has been proposed to be an inhibitory process in the metal-centered hydrolysis of peptide bonds by astacin and serralysin (a bacterial metalloprotease containing an astacin-like active site) (24,25). A metal-centered mechanism has been proposed for astacin and serralysin based on kinetic and spectroscopic studies of the enzymes and their Cu 2ϩ derivatives (24,25).…”

“…The status of the coordinated tyrosine has been proposed to be an inhibitory process in the metal-centered hydrolysis of peptide bonds by astacin and serralysin (a bacterial metalloprotease containing an astacin-like active site) (24,25). A metal-centered mechanism has been proposed for astacin and serralysin based on kinetic and spectroscopic studies of the enzymes and their Cu 2ϩ derivatives (24,25). In this mechanism, the active site Zn 2ϩ coordinated by three His, a tyrosine, and a water molecule can be activated via detachment of the Tyr-phenolate with a concomitant deprotonation of the coordinated water assisted by a glutamate residue to afford the arrangement Zn 2ϩ -OH⅐⅐⅐Glu Ϫ without a coordinated Tyr.…”

Insect pathogenicity in plant-beneficial pseudomonads: phylogenetic distribution and comparative genomics

“…The gene aprA is present in all 25 genomes, except that of strain P1.8, whereas aprX was only detected in the genomes of strains belonging to sub-clade 1 (Supplementary Table S1). AprA and AprX belong to the M10 family that includes serralysin, aeruginolysin and other related exopeptidases that cause tissue damage and anaphylactic responses (Park and Ming, 2002). In Pseudomonas entomophila, an aprA mutant was shown to be slightly less virulent and to have a reduced persistence in D. melanogaster (Liehl et al, 2006).…”