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

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• 作者：Brian Bennett and Richard C. Holz
• 刊名：Biochemistry
• 出版年：1997
• 出版时间：August 12, 1997
• 年：1997
• 卷：36
• 期：32
• 页码：9837 - 9846
• 全文大小：279K
• 年卷期：v.36,no.32(August 12, 1997)
• ISSN：1520-4995

文摘

The Co(II)Zn(II)- andZn(II)Co(II)-substituted derivatives of theaminopeptidase fromAeromonas proteolytica (AAP) were probed by EPRspectroscopy. EPR spectra of the high-spin S =³/₂Co(II) ions in [CoZn(AAP)] and [ZnCo(AAP)] indicatedthat each metal binding site provides aspectroscopically distinct signature. For [CoZn(AAP)],subtraction of EPR spectra recorded at pH 7.5and 10 revealed that two species were present and that the relativecontributions to each of the experimentalspectra were pH-dependent. The first EPR species, predominant atlower pH values, was simulated as arelatively featureless axial signal with g_effvalues of 2.20, 3.92, and 5.23 which correspond to anM_s =±¹/₂> ground state transition with ag_real of 2.29 and an E/D of 0.1.The second species, predominant athigh pH, was simulated with g_eff values of 1.80,2.75, and 6.88 and exhibited a characteristic eight-line⁵⁹Co hyperfine pattern with anA_z(⁵⁹Co) 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 speciesprovided an excellent simulation of the observed [CoZn(AAP)] EPRspectrum. The EPR spectrum of[ZnCo(AAP)] also contained two species, at least one of whichalso exhibited ⁵⁹Co hyperfine features.However, this signal exhibited little pH dependence, andindividual species could not be isolated. Theaddition of the competitive inhibitor 1-butaneboronic acid (BuBA) to[CoZn(AAP)] resulted in a distinctchange in the EPR spectrum; however, addition of BuBA to[ZnCo(AAP)] left the EPR spectrum completelyunperturbed. These data indicate that BuBA binds only to the firstmetal binding site in AAP and doesnot interact with the second site. On the basis of the X-raycrystallographic data for the transition stateanalog-inhibited complexes of AAP and the aminopeptidase from bovinelens, BuBA was reclassified asa substrate analog inhibitor rather than a transition state analoginhibitor as previously suggested [Baker,J. O., & Prescott, J. M. (1983) Biochemistry 22,5322-5331]. From difference spectroscopy and fromthe simulation of the [CoZn(AAP)] EPR spectrum, a third signalappearing upon BuBA binding wasisolated. This signal was simulated withg_eff values of 2.08, 3.15, and 6.15 whichcorrespond to an M_s =±¹/₂> ground state transition with ag_real of 2.41 and an E/D of 0.22.This simulation also invoked aneight-line unresolved ⁵⁹Co hyperfine pattern with anA_z(⁵⁹Co) value of4.0 mT. Summation of the thesethree species provided an excellent simulation of the observed[CoZn(AAP)] + BuBA EPR spectrum atboth pH values. This work establishes that substrate binds only tothe first metal binding site in AAPand thus substantiates the first step in catalysis in the recentlyproposed mechanism of action for AAP[Bennett, B., & Holz, R. C. (1997) J. Am. Chem. Soc.119, 1923-1933; Chen, G., et al. (1997)Biochemistry36, 4278-4286].