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Titolo:
SPECTROSCOPICALLY DISTINCT COBALT(II) SITES IN HETERODIMETALLIC FORMSOF THE AMINOPEPTIDASE FROM AEROMONAS-PROTEOLYTICA - CHARACTERIZATION OF SUBSTRATE-BINDING
Autore:
BENNETT B; HOLZ RC;
Indirizzi:
UTAH STATE UNIV,DEPT CHEM & BIOCHEM LOGAN UT 84322 UTAH STATE UNIV,DEPT CHEM & BIOCHEM LOGAN UT 84322
Titolo Testata:
Biochemistry
fascicolo: 32, volume: 36, anno: 1997,
pagine: 9837 - 9846
SICI:
0006-2960(1997)36:32<9837:SDCSIH>2.0.ZU;2-H
Fonte:
ISI
Lingua:
ENG
Soggetto:
LENS LEUCINE AMINOPEPTIDASE; ELECTRON-PARAMAGNETIC-RESONANCE; PERIPLASMIC NITRATE REDUCTASE; PURPLE ACID-PHOSPHATASE; X-RAY STRUCTURE; CRYSTAL-STRUCTURE; ESCHERICHIA-COLI; ACTIVE-SITE; DNA-POLYMERASE; UTEROFERRIN;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Science Citation Index Expanded
Citazioni:
50
Recensione:
Indirizzi per estratti:
Citazione:
B. Bennett e R.C. Holz, "SPECTROSCOPICALLY DISTINCT COBALT(II) SITES IN HETERODIMETALLIC FORMSOF THE AMINOPEPTIDASE FROM AEROMONAS-PROTEOLYTICA - CHARACTERIZATION OF SUBSTRATE-BINDING", Biochemistry, 36(32), 1997, pp. 9837-9846

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 lowerpH values, was simulated as a relatively featureless axial signal with g(eff) values of 2.20, 3.92, and 5.23 which correspond to an M-s = \/-1/2] ground state transition with a g(real) of 2.29 and an E/D of 0.1. The second species, predominant at high pH, was simulated with g(eff) values of 1.80, 2.75, and 6.88 and exhibited a characteristic eight-line Co-59 hyperfine pattern with an A(z)(Co-59) of 7.0 mT. These parameters correspond to an M-s = \+/-1/2] ground state transition with a g(real) 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 Co-59 hyperfine features. However, this signal exhibited little pHdependence, 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 g(eff) values of 2.08, 3.15, and 6.15 which correspond toan M-s = \+/-1/2] ground state transition with a g(real) of 2.41 and an E/D of 0.22. This simulation also invoked an eight-line unresolved Co-59 hyperfine pattern with an A(z)(Co-59) value of 4.0 mT. Summationof 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 inAAP and thus substantiates the first step in catalysis in the recently proposed mechanism of action for AAP.

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Documento generato il 01/12/20 alle ore 19:52:37