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. 1972 Oct;69(10):2850–2854. doi: 10.1073/pnas.69.10.2850

Similarities Between the Conformation of Arsanilazotyrosine 248 of Carboxypeptidase Aα in the Crystalline State and in Solution

Florante A Quiocho 1, Cecil H Mcmurray 1, William N Lipscomb 1
PMCID: PMC389660  PMID: 4507609

Abstract

Modification of carboxypeptidase Aγ crystals (Anson) with diazotized arsanilic acid specifically labels tyrosine 248; at pH 8.2 the modified enzyme gives yellow crystals, but a red solution. It has been suggested that arsanilazotyrosine 248 forms a complex with the Zn cofactor accounting for the red color in solution, but that a complex is not formed in the crystal. However, the crystal structure of carboxypeptidase Aγ is unknown. We show here that crystals of carboxypeptidase Aα, whose crystal structure has been determined, are red both in solution and in the crystalline state (at pH 8.2) after modification with diazotized arsanilic acid. These new data are of importance in relating the structure in the crystalline state to the catalytic mechanisms, as based on the x-ray diffraction evidence.

The activity of carboxypeptidase A in the crystal and in solution has a ratio of only 1/3 for the α form, in contrast to the ratio of 1/300 for the γ form, with carbobenzoxyglycyl-L-phenylalanine as a substrate.

A pH-jump experiment monitored by stopped-flow kinetics in a split-beam apparatus has revealed a single exponential rate when a solution of arsanilazotyrosine 248 carboxypeptidase Aα at pH 6.7 (yellow) is increased to pH 8.5 (red). The rate constants obtained in this experiment are 6.1 sec-1 at 3.0 mg/ml and 7.2 sec-1 at 1.6 mg/ml concentration of enzyme.

Keywords: enzyme activity, stopped-flow pH-jump

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

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