Mechanism of action of zinc proteinases: A MNDO/d/H study of alternative general‐acid general‐base catalytic pathways for carboxypeptidase‐A

Kilshtain-Vardi, Alexandra; Shoham, G.; Goldblum, Amiram

doi:10.1002/qua.10094

Cited by 20 publications

(24 citation statements)

References 47 publications

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“…X-ray crystallography and kinetic studies of CPA, together with more advanced theoretical calculations, suggested a contradicting model in which the water molecule is bound to the zinc ion throughout its transformation to the hydroxyl ion. This process is then followed by the binding of the peptide carbonyl to the zinc ion to form a pentacovalent zinc-protein-substrate complex (3,4). Our results provide realtime and direct evidence for the formation of the pentacoordinate catalytic zinc-protein complex during the peptide hydrolysis reaction as proposed by Lipscomb and coworkers (3).…”

Section: Implications For Understanding Protease Reaction Mechanismssupporting

confidence: 74%

“…1a and SI Text). In this model, the zinc remains tetrahedrally coordinated throughout the reaction (4,14). X-ray crystallography and kinetic studies of CPA, together with more advanced theoretical calculations, suggested a contradicting model in which the water molecule is bound to the zinc ion throughout its transformation to the hydroxyl ion.…”

Section: Implications For Understanding Protease Reaction Mechanismsmentioning

confidence: 99%

“…1a) to catalyze the hydrolysis of peptide bonds in a wide variety of substrates in both normal and pathological processes (1,2). Over the last five decades, substantial efforts have been aimed at understanding the molecular basis by which the catalytic zinc machinery executes such enzymatic reactions (3,4). Protein crystallography, proteomic, and theoretical studies have been instrumental in proposing reaction mechanisms (3,5,6).…”

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confidence: 99%

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Key feature of the catalytic cycle of TNF-α converting enzyme involves communication between distal protein sites and the enzyme catalytic core

Solomon

Akabayov

Frenkel

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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Despite their key roles in many normal and pathological processes, the molecular details by which zinc-dependent proteases hydrolyze their physiological substrates remain elusive. Advanced theoretical analyses have suggested reaction models for which there is limited and controversial experimental evidence. Here we report the structure, chemistry and lifetime of transient metal-protein reaction intermediates evolving during the substrate turnover reaction of a metalloproteinase, the tumor necrosis factor-␣ converting enzyme (TACE). TACE controls multiple signal transduction pathways through the proteolytic release of the extracellular domain of a host of membrane-bound factors and receptors. Using stopped-flow x-ray spectroscopy methods together with transient kinetic analyses, we demonstrate that TACE's catalytic zinc ion undergoes dynamic charge transitions before substrate binding to the metal ion. This indicates previously undescribed communication pathways taking place between distal protein sites and the enzyme catalytic core. The observed charge transitions are synchronized with distinct phases in the reaction kinetics and changes in metal coordination chemistry mediated by the binding of the peptide substrate to the catalytic metal ion and product release. Here we report key local charge transitions critical for proteolysis as well as long sought evidence for the proposed reaction model of peptide hydrolysis. This study provides a general approach for gaining critical insights into the molecular basis of substrate recognition and turnover by zinc metalloproteinases that may be used for drug design.dynamics ͉ matrix metalloproteinases ͉ proteolysis ͉ x-ray absorption ͉ stopped flow Z inc-dependent metalloproteinases comprise a large superfamily of enzymes possessing key biological roles. These enzymes use zinc (Fig. 1a) to catalyze the hydrolysis of peptide bonds in a wide variety of substrates in both normal and pathological processes (1, 2). Over the last five decades, substantial efforts have been aimed at understanding the molecular basis by which the catalytic zinc machinery executes such enzymatic reactions (3, 4). Protein crystallography, proteomic, and theoretical studies have been instrumental in proposing reaction mechanisms (3,5,6). However, the molecular details that link the catalytic chemistry to key kinetic, electronic, and structural events have remained elusive because of the difficulties associated with probing time-dependent structure-function aspects of such reactions in the presence of peptide substrates.Here we used a strategy based on dynamic structural spectroscopy to elucidate in detail the molecular mechanisms at work during substrate turnover by TACE. TACE is a disintegrin and metalloproteinase family member (also known as ADAM 17) with key roles in the regulation of the proteolytic release of cytokines, chemokines, growth factors, and receptors from cellular membranes (a process known as ectodomain shedding) (7). We have used stopped-flow freeze-quench x-ray absorption spectros...

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Section: Implications For Understanding Protease Reaction Mechanismssupporting

confidence: 74%

Section: Implications For Understanding Protease Reaction Mechanismsmentioning

confidence: 99%

mentioning

confidence: 99%

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Key feature of the catalytic cycle of TNF-α converting enzyme involves communication between distal protein sites and the enzyme catalytic core

Solomon

Akabayov

Frenkel

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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“…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.…”

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confidence: 96%

“…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).…”

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confidence: 99%

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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Metalloproteinases, Biophysics and Chemistry of

Solomon

Selzer

Milla

et al. 2008

Wiley Encyclopedia of Chemical Biology

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Zinc‐dependent metalloproteases constitute a large family of enzymes, part of the proteinase superfamily. Fundamental to the structural integrity and catalytic activity of metalloproteases is the presence of both zinc and calcium ions in the structure of the protein. This article will focus on matrix metalloproteinases that are involved in extracellular matrix (ECM) catabolism and serve as important enzymes in many aspects of biology, ranging from cell proliferation, differentiation, and proliferation to cancer, tumor metastasis, inflammation, and other pathologic states. Despite their key role in many normal and pathologic processes, the molecular mechanisms by which zinc‐dependent proteases hydrolyze their physiologic substrates are only known partly. Recent theoretical analyses have suggested reaction models for which limited and controversial experimental evidence exists. Here we will discuss the importance of quantifying the biophysical properties and the structural dynamic behavior of these enzymes to reveal their underlying molecular mechanisms. Such molecular knowledge holds promise in providing the basis for the novel design of specific antagonists as drug candidates for these important enzymes. In addition, we will discuss the use of real‐time spectroscopic tools for studying the reactive metal sites in these enzymes.

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Mechanism of action of zinc proteinases: A MNDO/d/H study of alternative general‐acid general‐base catalytic pathways for carboxypeptidase‐A

Cited by 20 publications

References 47 publications

Key feature of the catalytic cycle of TNF-α converting enzyme involves communication between distal protein sites and the enzyme catalytic core

Key feature of the catalytic cycle of TNF-α converting enzyme involves communication between distal protein sites and the enzyme catalytic core

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

Metalloproteinases, Biophysics and Chemistry of

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