Refined structure of bovine carboxypeptidase A at 1.25 Å resolution

Kilshtain-Vardi, Alexandra; Glick, Meir; Greenblatt, H.M.; Goldblum, Amiram; Shoham, G.

doi:10.1107/s0907444902015706

Cited by 35 publications

(24 citation statements)

References 38 publications

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“…This comparison, in contrary to the recent ''final'' deduction of Ref. 28, already indicates at least different rate-determining steps for these substrates. However, let us consider other experimental results prior to drawing more decisive conclusions.…”

contrasting

confidence: 71%

“…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.…”

mentioning

confidence: 97%

“…A variety of catalytic mechanisms proposed for CPA so far can be grouped into two major patterns defined as the ''promoted water'' and ''anhydride intermediate'' pathways (vide infra). [4][5][6][12][13][14][15][16][20][21][22][23][24][25][26][27][28] Although, either of them can be attributed to both, the peptide and ester hydrolysis, because of the particular kinetic evidence accumulated so far, it is usually assumed that the first pattern is applicable for peptides, whilst another is effective for esters (see Results and Discussion). Indeed, the question regarding the diverse catalytic patterns of CPA for specific peptide and ester substrates appears to be the most conspicuous one.…”

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confidence: 99%

“…[1][2][3][4][5][6][7][8] Because of two major reasons: (a) it was among the first proteases to be discovered, 1,5 and (b) it represents a broad class of important zinc-containing enzymes, [9][10][11] CPA has long been considered as a model enzyme for extensive mechanistic studies. [1][2][3][4][5][6][7][8][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27] These studies encompassed standard and sophisticated structural, [6][7][8]28 kinetic, [1][2][3][4][5][12][13][14][15]…”

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confidence: 99%

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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

et al. 2011

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Catalytic mechanisms of carboxypeptidase A (CPA) are well known for their diversity and the relative inaccessibility for a decisive comprehension. Recent encouraging attempts through modern computational techniques promoted new challenges for the complementary experimental endeavors. In this work, we have applied the stopped-flow technique and the method of reaction progress curve fitting to extract kinetic parameters for the CPA-catalyzed hydrolyses of smaller (typical) peptide and ester substrates, known for their strong activating/inhibiting impact, thus to which the traditional method of "initial rates" is not applicable. Our approach that innately implies the overall constancy of the affecter (substrate plus "active" product) concentration, made it possible to rigorously determine the physically meaningful "effective" values for the catalytic and Michaelis constants under diverse experimental conditions including variable temperature and urea or trimethylamine N-oxide concentrations. Analysis of the obtained results allowed for: (i) the further substantiation of diverse mechanistic patterns for archetypal specific peptide and ester substrates, (ii) testing and disclosure of intrinsic links between the stabilizing/destabilizing and activating/inhibiting effects for the important model enzyme, CPA, and (iii) tentative explanation of a distinct activating/inhibiting impact of these substrates through the strong specific interaction of their benzyl (Bz) moiety with the substrate binding S(3) subsite of CPA. We have demonstrated that stabilization of CPA either through the interaction with an extra Bz moiety (belonging to another substrate or to the product) leads to the increase of its catalytic power with respect to the specific peptide substrate and to its decrease with respect to the counterpart ester substrate. We conjecture that the catalytic mechanisms operating in these two cases include: (a) the "promoted water" mechanism for the peptide substrate that, seemingly, provides the almost "perfect induced fit" (low-barrier conformational adaptation), and (b) presumably, the "anhydride intermediate" mechanism for the ester substrate that, anyway, requires substantial conformational rearrangement (in fact, "partial or local unfolding") of the protein environment in the course of the rate-determining step.

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confidence: 71%

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confidence: 97%

mentioning

confidence: 99%

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confidence: 99%

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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

et al. 2011

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“…A higher resolution crystal structure of CPA suggests that the zinc-bound water is in its neutral state. 40 Several computational works, based on small cluster models and sometimes semi-empirical methods, have shed light on the mechanism of peptide hydrolysis in CPA. [41][42][43][44] The water-promoted mechanism, schematically depicted in Fig.…”

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confidence: 99%