Complex between carboxypeptidase A and a hydrated ketomethylene substrate analogue.

Shoham, G.; Christianson, D.W.; Oren, Deena A.

doi:10.1073/pnas.85.3.684

Cited by 35 publications

(28 citation statements)

References 29 publications

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“…Most likely the conformational flexibility of these residues is related to the two states of the active site; the free state and that with the substrate bound. Comparison of the present CPT structure with those of native CPA (Rees et al, 1983) and of CPA complexes (Rees et al, 1981 ;Shoham et al, 1988;Kim and Lipscomb, 1990;Kim and Lipscomb, 1991) suggests that the active site in CPT resembles the 'bound' state. Although the conformational changes observcd for Argl27 and Arg145 are not so pronounced as for Tyr248, they are clear enough to be described as changes in the hydrogenbonding network (here and in the following paragraph, the CPA numbering is used).…”

Section: T M P N G F a F T H S E --N R L W R K T R S V T S S S L C V mentioning

confidence: 97%

Crystal structure of carboxypeptidase T from Thermoactinomyces vulgaris

Teplyakov

Polyakov

Obmolova

et al. 1992

European Journal of Biochemistry

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The crystal structure of carboxypeptidase T from Thermoactirzomyces vulgaris has been determined at 0.235-nm resolution by X-ray diffraction. Carboxypeptidase T is a remote homologue of mammalian Zn-carboxypeptidases. In spite of the low degree of amino acid sequence identity, the threedimensional structure of carboxypeptidase T is very similar to that of pancreatic carboxypeptidases A and B. The core of the protein molecule is formed by an eight-stranded mixed p sheet. The active site is located at the C-edge of the central (parallel) part of the B sheet. The structural organization of the active centre appears to be essentially the same in the three carboxypeptidases. Amino acid residues directly involved in catalysis and binding of the C-terminal carboxyl of a substrate are strictly conserved. This suggests that the catalytic mechanism proposed for the pancreatic enzymes is applicable to carboxypeptidase T and to the whole family of Zn-carboxypeptidases. Comparison of the amino acid replacements at the primary specificity pocket of carboxypeptidases A, B and T provides an explanation of the unusual 'A+B' type of specificity of carboxypeptidase T. Four calcium-binding sites localized in the crystal structure of carboxypeptidase T could account for the high thermostability of the protein.Metallocarboxypeptidases are exopeptidases which contain a zinc ion in the active centre and catalyze the hydrolysis of C-terminal amino acids from polypeptide substrates. Extensive studies of bovine pancreatic carboxypeptidase A (CPA) by different techniques including crystallography, spectroscopy, kinetics and site-directed mutagenesis resulted in the understanding of the general principles of the catalytic mechanism (Christianson and Lipscomb, 1989). Three-dimensional structures have been determined for pancreatic carboxypeptidases A (Rees et al., 1983;Kobe and Goldsmith, 1990) and B (Schmid and Herriott, 1976; Coll et al., 1991) which share a common polypeptide fold but differ in substrate specificity (Hartsuck and Lipscomb, 1971 ;Folk, 1971 polypeptide chain of 326 amino acids and contains one zinc ion (Osterman et al., 1984). The amino acid sequence of CPT, deduced from the gene encoding the protein (Smulevitch et al., 1991), shows only moderate similarity with the CPA and CPB sequences; 30% and 27% identical residues, respectively. CPT exhibits an unusual 'dual'-substrate specificity combining features of both CPA and CPB; it is able to split off hydrophobic and basic amino acids with comparable efficiency (Osterman et al., 1984). As other proteins from Thermoactinomyces, CPT has an increased thermal stability in the presence of calcium ions (Osterman et al., 1984).Structure/functional studies of CPT provide a possibility to study the general principles of catalysis by Zn-carboxypeptidases. On the basis of the comparative primary-structure analysis, all Zn-carboxypeptidases were divided into two groups ; ,,digestive,, and ,,regulatory,, enzymes (Osterman et al., 1992). Pancreatic CPA and CPB appear to be structurally ...

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Section: T M P N G F a F T H S E --N R L W R K T R S V T S S S L C V mentioning

confidence: 97%

Crystal structure of carboxypeptidase T from Thermoactinomyces vulgaris

Teplyakov

Polyakov

Obmolova

et al. 1992

European Journal of Biochemistry

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“…CPA was purchased from Sigma Co.; the protein was further puri®ed and crystallized according to previously described procedures (Shoham et al, 1984(Shoham et al, , 1988. The fully grown crystals of native CPA were cross-linked prior to X-ray data measurement in order to impart greater mechanical strength.…”

Section: Crystallization and Data Collectionmentioning

confidence: 99%

“…CPA has been the subject of extensive research in the last 30 years (Coleman & Vallee, 1961;Neurath et al, 1970;Shoham et al, 1984Shoham et al, , 1988Christianson & Lipscomb, 1989;Feinberg et al, 1993Feinberg et al, , 1995Greenblatt et al, 1998). However, the exact catalytic mode of action of this representative enzyme and the rest of the zinc proteinase family is still not completely clari®ed.…”

Section: Introductionmentioning

confidence: 99%

Refined structure of bovine carboxypeptidase A at 1.25 Å resolution

Kilshtain-Vardi

Glick

Greenblatt

et al. 2003

Acta Crystallogr D Biol Cryst

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The crystal structure of the bovine zinc metalloproteinase carboxypeptidase A (CPA) has been refined to 1.25 A resolution based on room-temperature X-ray synchrotron data. The significantly improved structure of CPA at this resolution (anisotropic temperature factors, R factor = 10.4%, R(free) = 14.5%) allowed the modelling of conformational disorders of side chains, improved the description of the protein solvent network (375 water molecules) and provided a more accurate picture of the interactions between the active-site zinc and its ligands. The calculation of standard uncertainties in individual atom positions of the refined model of CPA allowed the deduction of the protonation state of some key residues in the active site and confirmed that Glu72 and Glu270 are negatively charged in the resting state of the enzyme at pH 7.5. These results were further validated by theoretical calculations that showed significant reduction of the pK(a) of these side chains relative to solution values. The distance between the zinc-bound solvent molecule and the metal ion is strongly suggestive of a neutral water molecule and not a hydroxide ion in the resting state of the enzyme. These findings could support both the general acid/general base mechanism, as well as the anhydride mechanism suggested for CPA.

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“…It was assumed that the two tetrahedral oxygens of the gem-diol are stabilized by their interactions with the active site. That is, the gem-diol state is highly unstable in solution, unless strong electron attractors are present near the carbonyl 50. It was concluded that the gem-diol forms only as a result of interaction with the enzyme, while none could be detected in solution with no enzyme present 50.…”

Section: Introductionmentioning

confidence: 99%

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

Kilshtain

Warshel

2009

Proteins

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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.

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Complex between carboxypeptidase A and a hydrated ketomethylene substrate analogue.

Cited by 35 publications

References 29 publications

Crystal structure of carboxypeptidase T from Thermoactinomyces vulgaris

Crystal structure of carboxypeptidase T from Thermoactinomyces vulgaris

Refined structure of bovine carboxypeptidase A at 1.25 Å resolution

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

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