Title

Spectroscopically Distinct Cobalt(II) Sites in Heterodimetallic Forms of the Aminopeptidase from Aeromonas proteolytica:  Characterization of Substrate Binding

Document Type

Article

Language

eng

Format of Original

10 p.

Publication Date

8-12-1997

Publisher

American Chemical Society

Source Publication

Biochemistry

Source ISSN

0006-2960

Original Item ID

doi: 10.1021/bi970735p

Abstract

The Co(II)Zn(II)- and Zn(II)Co(II)-substituted derivatives of the aminopeptidase from Aeromonas proteolytica (AAP) were probed by EPR spectroscopy. EPR spectra of the high-spin S = 3/2 Co(II) ions in [CoZn(AAP)] and [ZnCo(AAP)] indicated that each metal binding site provides a spectroscopically distinct signature. For [CoZn(AAP)], subtraction of EPR spectra recorded at pH 7.5 and 10 revealed that two species were present and that the relative contributions to each of the experimental spectra were pH-dependent. The first EPR species, predominant at lower pH values, was simulated as a relatively featureless axial signal with geff values of 2.20, 3.92, and 5.23 which correspond to an Ms = |±1/2〉 ground state transition with a greal of 2.29 and an E/D of 0.1. The second species, predominant at high pH, was simulated with geff values of 1.80, 2.75, and 6.88 and exhibited a characteristic eight-line 59Co hyperfine pattern with an Az(59Co) of 7.0 mT. These parameters correspond to an Ms = |±1/2〉 ground state transition with a greal of 2.54; however, the signal exhibited marked rhombicity (E/D = 0.32) indicative of an asymmetric tetrahedral or five-coordinate Co(II) ion. Summation of these two species provided an excellent simulation of the observed [CoZn(AAP)] EPR spectrum. The EPR spectrum of [ZnCo(AAP)] also contained two species, at least one of which also exhibited 59Co hyperfine features. However, this signal exhibited little pH dependence, and individual species could not be isolated. The addition of the competitive inhibitor 1-butaneboronic acid (BuBA) to [CoZn(AAP)] resulted in a distinct change in the EPR spectrum; however, addition of BuBA to [ZnCo(AAP)] left the EPR spectrum completely unperturbed. These data indicate that BuBA binds only to the first metal binding site in AAP and does not interact with the second site. On the basis of the X-ray crystallographic data for the transition state analog-inhibited complexes of AAP and the aminopeptidase from bovine lens, BuBA was reclassified as a substrate analog inhibitor rather than a transition state analog inhibitor as previously suggested [Baker, J. O., & Prescott, J. M. (1983) Biochemistry 22, 5322−5331]. From difference spectroscopy and from the simulation of the [CoZn(AAP)] EPR spectrum, a third signal appearing upon BuBA binding was isolated. This signal was simulated with geff values of 2.08, 3.15, and 6.15 which correspond to an Ms = |±1/2〉 ground state transition with a greal of 2.41 and an E/D of 0.22. This simulation also invoked an eight-line unresolved 59Co hyperfine pattern with an Az(59Co) value of 4.0 mT. Summation of the these three species provided an excellent simulation of the observed [CoZn(AAP)] + BuBA EPR spectrum at both pH values. This work establishes that substrate binds only to the first metal binding site in AAP and thus substantiates the first step in catalysis in the recently proposed mechanism of action for AAP [Bennett, B., & Holz, R. C. (1997) J. Am. Chem. Soc. 119, 1923−1933; Chen, G., et al. (1997) Biochemistry 36, 4278−4286].

Comments

Biochemistry, Vol. 36, No. 32 (August 12, 1997): 9837-9846. DOI.

Brian Bennett and Richard C. Holz were affiliated with Utah State University at the time of publication.