¹⁵N Nuclear Magnetic Resonance Studies of the Active Sites of Enzymes and In Vivo Nitrogen Metabolism

Author: Kanamori, Keiko

Year: 1981

Degree: Dissertation (Ph.D.)

Advisor: Roberts, John D.

Committee Member: Unknown, Unknown

Option: Chemistry

DOI: 10.7907/ryxe-9852

Abstract

To determine whether the catalytic zinc of carboxypeptidase A (CPA) is complexed to the active-site tyrosine (Tyr-248) in solution, a 15N probe, an arsanilazo group enriched in 15N at both azo nitrogens, has been selectively coupled to Tyr-248 with retention of full catalytic activity, and the resulting arsanilazotyrosyl-248 carboxypeptidase A (Zn.Azo-CPA) studied by 15N NMR spectroscopy over pH range from 7.0 to 10.3. Its azo nitrogen resonances, Na (proximal to Tyr-248) and NB (distal), show 8.3 ppm and 23.5 ppm shielding respectively relative to the corresponding resonances of apoarsanilazotyrosyl-248 carboxypeptidase A at pH 8.8. By comparison with the 15N shifts of a model azotyrosine and its zinc complex, these shift differences indicate substantial complexation of the azotyrosyl-248 with the catalytic zinc at pH 8.8. Consistent with such complexation is the fact that the azo nitrogen resonances of Zn.Azo-CPA show large deshieldings on addition of a slowly hydrolyzing substrate glycyl-L-tyrosine or an inhibitor β -phenylpropionate, each of which binds to the catalytic zinc and therefore is expected to break any azotyrosine-Zn complex. The results demonstrate unequivocally that the molecular basis for the properties of Zn.Azo-CPA observed by other spectral techniques is a Zn-azotyrosyl-248 complex, with the Nβ nitrogen (and presumably the phenolic oxygen) as ligand to zinc. Furthermore, the high sensitivity of 15N shifts to coordination with zinc permits at least a semi-quantitative estimate of the degree of azotyrosine-Zn complexation and indicates that the major conformation of ζ n.Azo-CPA, and by implication, of CPA in solution could be the one in which the active-site tyrosine is complexed to zinc, in contrast to the major conformation in the crystalline enzyme in which the Tyr-248 hydroxyl is 17 Å from the zinc.

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