V. alginolyticus PepD, which has been identified as a member of the metallopeptidase M20 family and is considered an aminoacylhistidine dipeptidase, was investigated. The putative active site of V. alginolyticus PepD could be proposed on the basis of sequence analysis and from the modeling results. We have successfully expressed and purified wild-type PepD and mutants from V. alginolyticus. The way V. alginolyticus PepD hydrolyzes
L-carnosine (β-Ala-L-His) is similar to how other known PepD do this.
Asp82 is two residues downstream from His80, in the vicinity of the zinc center, and is thought to clamp the imidazolium ring of His80 Nδ1. Additionally, potential hydrogen-bonding interactions of the Nδ1 porton of His80 with a side chain oxygen of Asp82 forming an Asp-His-Zn1 triad that has been postulated to decrease the Lewis acidity of Zn2+
and may further assist in facilitating the coordination of a double-bonded oxygen to Zn1(Fig.17 step 0 - 2). A catalytic mechanism is proposed that the bridging catalytic water attacks the carbonyl carbon of the scissile peptide bond to form a sp3-orbital substrate-enzyme tetrahedral intermediate (Fig.17 step 3 - 4). Therefore, not only does the Zn2 coordinated by the carboxylate oxygen of Glu150 cause these Glu150 mutants to lose enzyme activity, but the Asp82 mutants also experience lost enzyme activity, suggesting that the probable role of Asp82 on PepD is clamping the imidazolium ring of His80 Nδ1, so as to correctly coordinate Zn1 to enhance substrate and metal binding ability. The substitution of Glu150 to Asp was resulted in only partial loss of the enzymatic activity; maybe the replacement of Glu with Asp at this position only partially affects the metal ligand-binding affinity and subsequent activation of the catalytic water for substrate-enzyme tetrahedral intermediate formation.
Fig. 17: Proposed mechanism for the hydrolysis of PepD, catalyzed by a metallopeptidase with a co-catalytic active site, where R1, R2 and R3 are substrate side chains, and R is an N-terminal amine or a C-terminal carboxylate. This mechanism is based upon the proposed mechanism for the aminopeptidase from Aeromonas proteolytica.
On the basis of structural model superposition of PepD on PepV after optimal fit, the substrate binding site residues and the metal center catalytic domain both are shown to be important. After mutation analysis of several residues, only Arg369 exhibited reduced enzymatic activity to hydrolyze L-carnosine as a substrate. The role of Arg369 not only is located at the probable dimerization domain, but it also appears to be that part of the lid domain, at which the substrate carboxyl end should be trapped in the hydrogen bond network dominated by the Arg369 guanidyl group, that contributes to the tightening of substrate
binding (Fig.17 step 1). Unfortunately, the mutant PepD of the other three residues did not lose all of catalytic activity, as expeced; and that His219Ala increased enzymatic activity was a surprise. The enzyme kinetics study showed that the relative activities of His219Ala mutant protein in terms of kcat and kcat/Km were better then wild-type. Moreover, the Km value of Asn260Ala suggested the mutant of this residue was influencing on substrate binding ability, and the shifted kcat value of Gly435Ala indicate that was influencing on catalytic activity.
Maybe the key conclusions that can be drawn from this study are that these residues need to be studied in greater depth.
V. alginolyticus PepD is a 54kD metallopeptidase, which is activated by Zn2+ in its wild-type. The functional importance of metal ions - including Co2+, Cu2+, Ni2+, Mg2+, Mn2+
and Cd2+ - may be indicated by the different levels of restored activity and the apparent value shift to kcat in enzyme kinetics of the reconstituted metal PepD protein. Possible roles for both metal ions in PepD that contains co-catalytic active sites include: (i) binding and positioning substrate; (ii) binding and activating a water molecule to yield an active site hydroxide nucleophile; and (iii) stabilizing the transition state of a hydrolytic reaction. The Lewis acidity of the metal ions is the key factor in metal-centered hydrolysis, in which the pKa of the coordinated water is significantly lowered to assist its nucleophilic attack on the scissile bond at a neutral pH. Consistent with this hypothesis, both Mg2+ and Mn2+ ions bind to the active site of APPro in a very similar resule, which Mn2+ activates APPro and Mg2+ does not [49]. Mg2+ has a higher charge density and is a harder Lewis acid than any other metal ion that is widely available in biological systems. This property makes Mg2+ the perfect metal ion for binding to the hard oxygen anions of the negatively-charged phosphodiester backbone of nucleic acids. As a result, Mg2+ is a good key factor for nucleases and other phosphohydrolases, but not for peptidase. Zn2+, Mn2+ and Co2+ have a lower charge density than Mg2+, and are stronger Lewis acids, based upon their respective ionization potentials.
Perhaps that is the reason for nature’s apparent choice of Zn2+, Mn2+ or Co2+ at the catalytically-active site for most peptidases.
In conclusion, V. alginolyticus PepD is an aminoacylhistidine dipeptidase that can hydrolyze Xaa-His dipeptides, including an unusual dipeptide - carnosine (β-Ala-L-His). To confirm this, further investigations are warranted into the putative importance of functional residues that almost exhibit a decrease or lose of enzymatic activity. Our results reveal that these residues are involved in both substrate and metal binding, and that this dramatically affects enzymatic activity. The addition of metal ions led to the alteration in the specific activity of recombinant PepD and the activity is increased by metal ions that complex with PepD , like Mn2+, Co2+, Ni2+ and Cd2+. The improvement of activity and stability by modification of metal species would be promising from an aspect of the practical application of the enzyme.