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. 1973 Dec;70(12 Pt 1-2):3797–3801. doi: 10.1073/pnas.70.12.3797

Enzymatic Activities of Carboxypeptidase A's in Solution and in Crystals

William N Lipscomb 1
PMCID: PMC427331  PMID: 4521205

Abstract

Activities of all known forms of bovine carboxypeptidase A's (α having 307, β having 305, and γ having 300 amino acids) are essentially the same in solution under given conditions. However, activities in the crystals differ. The Aα crystals elongated along the a axis (a) have unit cell parameters a = 51.41 Å, b = 59.89 Å, c = 47.19 Å, and β = 97°35′, (b) show about [unk] of the activity of the dissolved enzyme, and (c) have the same color of the arsanilazo Tyr 248 derivative in the crystalline and solution states, namely red at pH 8.2 and yellow at pH 7.4. The Aγ crystals elongated along the b axis (a) have unit cell parameters a = 50.9 Å, b = 57.9 Å, c = 45.0 Å, and β = 94°40′, (b) show [unk] of the activity of the dissolved enzyme, and (c) show, in the arsanilazo Tyr 248 derivative, yellow crystals and red solution at pH 8.2.

Although the detailed three-dimensional structure is known for the Aα form described above, the structure of the Aγ form is as yet undertermined. A reasonable hypothesis is that the major part of the differences in crystal behaviors is due to differences in intermolecular (crystal-packing) interactions. In particular the movement of Tyr 248 may be somewhat hindered by these intermolecular contacts in the Aγ crystals, and in other crystalline forms which are elongated along the b axis. The movement observed in the x-ray diffraction studies, of the OH group of Tyr 248 by 12 Å when the very slowly hydrolyzed substrate Gly-Tyr is bound to Aα crystals, appears to be largely unhindered by intermolecular interactions.

Examination of a three-dimensional space-filling structural model of the carboxypeptidase A molecule reveals that the phenolic oxygen of Tyr 248 can approach within 2 Å of the Zn cofactor. This approach requires a movement by about 6 Å of the polypeptide chain in the general region of Tyr 248. Moreover, the position of Tyr 248 when bonded to Zn can just be seen in the electron density map of the crystal structure at a level which, averaged over many unit cells, suggests some 15-25% of the enzyme is in this form at pH 7.4 and 4° in the crystals of the x-ray diffraction study. It is probable that when the Zn-Tyr 248 bond is present the enzyme is catalytically inactive.

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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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