On the origin of the catalytic power of carboxypeptidase A and other metalloenzymes

Abstract

Zinc metalloenzymes play a major role in key biological processes and Carboxypeptidase-A (CPA) is a major prototype of such enzymes. The present work quantifies the energetics of the catalytic reaction of CPA and its mutants using the EVB approach. The simulations allow us to quantify the origin of the catalytic power of this enzyme and to examine different mechanistic alternatives. The first step of the analysis used experimental information to determine the activation energy of each assumed mechanism of the reference reaction without the enzyme. The next step of the analysis involved EVB simulations of the reference reaction and then a calibration of the simulations by forcing them to reproduce the energetics of the reference reaction, in each assumed mechanism. The calibrated EVB was then used in systematic simulations of the catalytic reaction in the protein environment, without changing any parameter. The simulations reproduced the observed rate enhancement in two feasible general acid-general base mechanisms (GAGB-1 and GAGB-2), although the calculations with the GAGB-2 mechanism underestimated the catalytic effect in some treatments. We also reproduced the catalytic effect in the R127A mutant. The mutation calculations indicate that the GAGB-2 mechanism is significantly less likely than the GAGB-1 mechanism. It is also found, that the enzyme loses all its catalytic effect without the metal. This and earlier studies show that the catalytic effect of the metal is not some constant electrostatic effect, that can be assessed from gas phase studies, but a reflection of the dielectric effect of the specific environment.

Diverse role of conformational dynamics in carboxypeptidase A‐driven peptide and ester hydrolyses: Disclosing the “Perfect Induced Fit” and “Protein Local Unfolding” pathways by altering protein stability

“…If the catalytic efficiency in this case is close to the optimal, it can be defined as an ''ideal induced fit.'' According to Warshel et al, 24,[94][95][96] ideal enzymatic catalysis should not require a large contribution from the conformational degrees of freedom, which means that the protein conformation remains within ''tier 2'' during the catalytic chemical process (see also Refs. 34,95).…”

“…As already mentioned in the Introduction, Bz-Gly-Phe and Bz-Gly-OPhe are prototype model substrates for CPA and have long been used for mechanistic studies using both, experimental 4,12,[17][18][19]22,45 and computational [23][24][25][26][27] techniques. Although a current outline of the previous experimental work implies two distinct mechanisms for typical (and specific) peptide and ester substrates such are Bz-Gly-Phe and Bz-Gly-OPhe (vide supra), recent, most sophisticated computational studies 26,27 deduced that not only the same overall mechanism (the ''promoted water'' pathway) is operative in both cases but also the rate-determining step, that is the nucleophilic attack of a scissile bond by water, accompanied by the concerted proton transfer to Glu270 (general base catalysis), is the same.…”

“…These include a cofactor, the divalent zinc ion (Zn 21 ), and a number of specific side groups of the Glu270, Tyr248, Ser197, Asn144, Arg145, Arg127, and Arg71 residues. 3,6,8,[23][24][25][26][27][28] This situation is rather distinct from some other classes of model enzymes such as, e.g., serine hydrolases for which the major mechanistic pattern has been firmly established. 29,30 Among other complexities, extremely diverse kinetic manifestations showing up through the pH profiles, [3][4][5][6]12 inhibition/activation patterns (due to substrates or other effectors), [3][4][5][6]12,13,22 cryospectroscopic (intermediate trapping) experiments, 6,14,16 and viscosity impact observations, [17][18][19] should be mentioned.…”

“…Interestingly, rather diverse performance has been observed not only between two families of more natural yet synthetic (''probing'') peptide and the counterpart ester (depsi-peptide) substrates but also within the substrates of either of these types that may differ in the chain length and/or residue specificity. [2][3][4][5][6]12,13 The emergence of rapidly progressing powerful computational techniques and two decades of respective serious attempts [23][24][25][26][27] shed some new light on this problem. However, the situation remains in major unclear and definitely requires involvement of additional counterbalancing experimental efforts (vide infra).…”

“…[3][4][5][6]12,13 These effects disappear for larger substrates (comprising polypeptide fragments with a peptide bond number more than 2), 3,12,13 which, however, are rarely available commercially and whose synthesis is a labor-and time-consuming procedure. In addition, even most sophisticated computational methods, in seeking for the CPA mechanisms, currently deal with smaller substrates, [23][24][25][26][27] which allow essential saving of computing resources. E.g., the latest most advanced computer simulations of CPA patterns were performed with the involvement of counterpart peptide and ester substrates, hippuryl-L-phenylalanine (Bz-Gly-Phe) and hippuryl-L-bpheyllactic acid (Bz-Gly-OPhe).…”