Kinetic properties of crystalline enzymes. Carboxypeptidase A

Spilburg, Curtis A.; Bethune, J. L.; Valee, B L

doi:10.1021/bi00625a018

Cited by 51 publications

(46 citation statements)

References 32 publications

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“…Spectroscopic studies of arsanilazotyrosine-248 CPase A (AAT-CPase A), however, have been interpreted as suggesting that a tyrosine-248-Zn interaction is an essential prerequisite for catalysis (6,(18)(19)(20)(21)(22), in direct contradiction to the conclusion from the crystallographic studies. Although AAT may chelate to the Zn in unliganded AAT-CPase A, it seems likely that this interaction is a property of the arsanilazo modification.…”

Section: Resultsmentioning

confidence: 98%

Binding of ligands to the active site of carboxypeptidase A.

Rees¹,

Lipscomb²

1981

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

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We compare the detailed binding modes of the 39-amino acid inhibitor from potatoes, glycyl-L-tyrosine, the ester analogue CH30C6H4(CO)CH2CH(CO0)C6H5, and indole acetate to the exopeptidase carboxypeptidase A (EC 3.4.17.1). In the potato inhibitor, cleavage of the COOH-terminal glycine-39 leaves a new carboxylate anion ofvaline-38 having one oxygen on zinc and the other as a receptor of a hydrogen bond from tyrosine-248 of carboxypeptidase. Tyrosine-248 also receives a hydrogen bond from the amide proton of the originally penultimate peptide bond between tyrosine-37 and valine-38. This hydrogen bond suggests product stabilization which is available to peptides and depsipeptides but not to esters lacking an equivalent peptide bond (nonspecific esters). Also, this structure may represent the intermediate binding step for the uncleaved substrate as it moves along the binding subsites. In particular, this may be the binding mode for the substrate after association of the COOH-terminal region of the substrate with the residues at binding subsite S2 (tyrosine-198, phenylalanine-279, and arginine-71) and preceding entry into the catalytic site S;. These stabilized complexes allow some understanding of the effect of indole acetate, shown here to bind in the pocket at S' , as a competitive inhibitor for esters (for which entry into S' precedes the rate-determining catalytic step for hydrolysis) and as a noncompetitive inhibitor for peptides (for which entry into S' is rate limiting). These results, including the binding mode of the ester analogue, are consistent with the original proposal from x-ray studies that both esters and peptides are cleaved with the carboxy terminus at S';, although not necessarily by the same chemical steps.

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Section: Resultsmentioning

confidence: 98%

Binding of ligands to the active site of carboxypeptidase A.

Rees¹,

Lipscomb²

1981

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

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“…In addition, the structure of another crystal form of CPase A should be of great relevance in resolving the controversy concerning the integrity of the molecular structure in solution as compared with the structure in the various crystalline phases. In particular, the conclusions of the x-ray diffraction studies of CPase A have been questioned (7,8) on the basis of the low reactivity (1/300) towards small substrates of one crystalline phase, even though the x-ray diffraction studies were made on a different crystal form that has an activity of about < Glu -Gln-His-Ala-Asp-Pro-Ile- 8 12 18…”

mentioning

confidence: 99%

Structure of the potato inhibitor complex of carboxypeptidase A at 2.5-A resolution.

Rees¹,

Lipscomb²

1980

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

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The structure of the complex between the proteolytic enzyme carboxypeptidase A (peptidyl-L-amino-acid hydrolase, EC 3.4.17.1) and the 39-amino-acid carboxypeptidase A inhibitor from potatoes has been determined at 2.5-A resolution. A combination of multiple isomorphous replacement, molecular replacement, and noncrystallographic symmetry averaging techniques was used to solve the structure. The chain trace of the inhibitor and details of the binding interactions in the complex are described. A surprising aspect of the complex is that the carboxy-terminal peptide bond of the inhibitor has been hydrolyzed, and the carboxy-terminal glycine is trapped in the binding pocket of carboxypeptidase A. Consequently, the complex resembles a stage in the catalytic mechanism after hydrolysis of the peptide bond. The ring of tyrosine-248, which is known to undergo large conformational changes upon substrate binding, is in the "down" position and interacts with the inhibitor in the complex.

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“…and K, values [31]. Interestingly, S. solfataricus carboxypeptidase displayed a k,,, for the hydrolysis of Cbz-Gly-Gly-Phe over tenfold higher than that determined for Cbz-Phe.…”

Section: Substrate Specificitymentioning

confidence: 74%

Purification and characterization of a thermostable carboxypeptidase from the extreme thermophilic archaebacterium Sulfolobus solfataricus

Colombo

D’Auria

Fusi

et al. 1992

European Journal of Biochemistry

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A carboxypeptidase was purified to electrophoretic homogeneity from the thermoacidophilic archaebacterium Sulfolobus solfataricus. Molecular masses assessed by SDS/PAGE and gel filtration were 42 kDa and 170 kDa, respectively, which points to a tetrameric structure for the molecule. An isoelectric point of 5.9 was also determined. The enzyme was proven to be a metalloprotease, as shown by the inhibitory effects exerted by EDTA and o-phenanthroline; furthermore, dialysis against EDTA led to a complete loss of activity, which could be restored by addition of Zn2+ in the micromolar range, and, to a lesser extent, by Co2+. The enzyme was endowed with a broad substrate specificity, as shown by its ability to release basic, acidic and aromatic amino acids from the respective benzoylglycylated and benzyloxycarbonylated amino acids. An esterase activity of the carboxypeptidase was also demonstrated on different esterified amino acids and dipeptides blocked at the N-terminus. The enzyme displayed broad pH optima ranging over 5.5-7.0, or 5.5-9.0, when using an acidic or a basic benzyloxycarbonylated amino acid, respectively. With regard to thermostability, it was proven to be completely stable on incubation for 15 min at 85°C. Furthermore, thanks to its relatively low activation energy, i.e. 31.0 kJ/mol, it was still significantly active at room temperature. At 40°C, the enzyme could withstand 0.1% SDS and different organic solvents: particularly ethanol up to 99%. Amino acid and N-terminal sequence analyses did not evidence any similarity to carboxypeptidases A nor thermolysin. A weak similarity was only found with bovine carboxypeptidase B.

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Kinetic properties of crystalline enzymes. Carboxypeptidase A

Cited by 51 publications

References 32 publications

Binding of ligands to the active site of carboxypeptidase A.

Binding of ligands to the active site of carboxypeptidase A.

Structure of the potato inhibitor complex of carboxypeptidase A at 2.5-A resolution.

Purification and characterization of a thermostable carboxypeptidase from the extreme thermophilic archaebacterium Sulfolobus solfataricus

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