Evidence of an essential histidine residue in thermolysin

Burstein, Yigal; Walsh, Kenneth A.; Neurath, Hans

doi:10.1021/bi00698a030

Cited by 217 publications

(126 citation statements)

References 17 publications

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“…Modification of lysyl residues with DEP cannot be reversed by hydroxylamine. Also, hydroxylamine at 20 mM can decarbethoxylate selectively histidyl residues under conditions that do not decarbethoxylate 2-O-carbethoxy tyrosyl residues [19]. In the present work, AP-P was completely inactivated at 0.2 mM DEP and could be reactivated by treatment with 20 mM NH2OH, implicating specific modification of histidyl residues.…”

Section: Discussionsupporting

confidence: 47%

Chemical modification of porcine kidney aminopeptidase P indicates the involvement of two critical histidine residues

Lim

Turner

1996

FEBS Letters

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Aminopeptidase P (AP-P), purified to homogeneity from porcine kidney membranes, was completely inactivated by treatment with 0.2 mM diethylpyrocarbonate (DEP) at pH 7.0. Treatment of the modified enzyme with 20 mM hydroxylamine resulted in recovery of AP-P activity. The differential absorption of native and modified AP-P at 240 nm showed that DEP modified two histidyl residues per mol of AP-P. The substrates, bradykinin(1-5) and Gly-Pro-Hyp, and also the inhibitor, apstatin, could protect against DEP inactivation. These results suggest that histidine residues are critical for AP-P activity.

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Section: Discussionsupporting

confidence: 47%

Chemical modification of porcine kidney aminopeptidase P indicates the involvement of two critical histidine residues

Lim

Turner

1996

FEBS Letters

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“…EFA can also react with tyrosyl residues in proteins, as revealed by a decrease in the difference absorption spectrum with a minimum at 278 nm [36]. No decrease in the absorption at 278 nm was, however, observed in any of the present fractions, indicating that no modification of tyrosyl residues had taken place during the incubation of b-cl complex with EFA.…”

Section: -510mentioning

confidence: 67%

Chemical modification studies of beef‐heart mitochondrial b‐c₁ complex

Lorusso

Gatti

Marzo

et al. 1987

European Journal of Biochemistry

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The effect of the histidine-modifier ethoxyformic anhydride (EFA) on the enzymatic properties of the mitochondrial b-el complex (ubiquinol -cytochrome c reductase) has been investigated. Chemical modification by EFA inhibited to the same extent the reductase and the proton translocating activity of the complex. In particular EFA modification of the complex resulted in: (a) strong inhibition of the antimycin-insensitive reduction of b cytochromes; (b) inhibition of the antimycin-promoted oxidant-induced reduction of b cytochromes and (c) inhibition of oxidation of pre-reduced b cytochromes.Analysis of the absorbance at 238 nm, indicative of N-(ethoxyformy1)histidine derivative, of the various polypeptide subunits separated by high-pressure liquid chromatography procedure, showed that EFA modified residues in core proteins and in the low-molecular-mass proteins. Both the inhibition of the redox and the protonmotive activity of the complex and the absorbance increase at 238 nm of the core protein fraction were readily reversed by hydroxylamine, indicating that modification of histidine residue(s) in core protein(s) is critical for the activity of the complex.This was supported by the finding that modification of the reductase with EFA prevented binding of fluoresceinisothiocyanate to histidine residue(s) in core protein 11. EFA modification of the reductase was without effect on the binding of N-(7-dimethylamino-4-methylcoumarinil)maleimide to the various polypeptides of the complex except for the binding to the Fe-S protein which was greatly potentiated. Thus primary chemical modification of histidine residue(s) in core protein (11) appears to cause, in turn, a conformational change in the Rieske Fe-S protein.The mitochondrial b-c, complex (ubiquinol -cytochrome c reductase) is a multisubunit enzyme which catalyzes the transfer of reducing equivalents from ubiquinol to cytochrome c. Electron flow in the reductase is linked to proton translocation both in the native and reconstituted membranes [l-71. This process results in the net transport of 1 H+/ealong with one positive charge from the inner to the outer space. In the latter, one additional H+/e-is released as the result of the scalar oxidation of an hydrogenated reductant by cytochrome c.SDS-PAGE analysis reveals in the protein complex not less than eight bands. The two high-molecular-mass bands represent core proteins I and 11, bands 3 -5 are apo-proteins bearing the metal redox centers, namely cytochromes b and c1 and the Rieske iron-sulphur protein [8 -lo]. The remaining subunits are low-molecular-mass polypeptides which migrate, depending on the experimental conditions, into three or five bands [8 -111.

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“…Ethoxyformic anhydride has also been reported as showing different group affinities in different proteins [16][17][18][19]231. In most proteins it acylates amino and/ or imidazole groups.…”

Section: Discussionmentioning

confidence: 99%