The mitochondrial processing peptidase behaves as a zinc-metallopeptidase.
The yeast mitochondrial processing peptidase (MPP) and its subunits were purified in Escherichia coli under conditions for which the enzyme retains most of its processing activity in the absence of externally added divalent cation. The holoenzyme exhibited a Km value of 1.35 microM and a Vmax value of 0.25 microM/min and was inhibited by metal chelators in a time-dependent manner. Measurement of the metal content showed that both, MPP and beta-MPP, contained 0.86 and 1.05 atoms of Zn2+ per molecule, respectively. An enzymatically inactive MPP mutant carrying a mutation of the first histidine of the putative metal-ion binding HXXEH motif in beta-MPP retained less than 0.2 atom of Zn2+ per molecule. A metal-free enzyme (apoenzyme) was prepared from the holoenzyme and shown to be devoid of any processing activity. Incubation of the apoenzyme with 50 nM and 500 nM Zn2+ restored 50% and 80% of the processing activity, respectively. However, no reactivation occurred at concentrations of Zn2+ higher than 1 microM. Addition of 500 nM Mn2+ or higher concentrations (up to 50 microM) reactivated only 50% of the processing activity. The holoenzyme was competitively inhibited by molar excess of Zn2+ (Ki of 3.1 microM) but not by molar excess of Mn2+. Taken together, our data suggest that the authentic MPP is a Zn2+ rather than a Mn2+ metallopeptidase.
Luciano
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P
Laboratoire d'Ingénierie des Systèmes Macromoléculaires, Institut de Biologie Structurale et Microbiologie, CNRS, 31 chemin Joseph Aiguier, 13402 Marseille, Cedex 20, France.
Tokatlidis
K
K
Chambre
I
I
Germanique
J C
JC
Géli
V
V
D004926:Escherichia coli / Q000235:genetics; D006868:Hydrolysis; D007700:Kinetics; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D011480:Protease Inhibitors / Q000494:pharmacology; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013379:Substrate Specificity; D015032:Zinc / Q000378:metabolism*; D000096122:Mitochondrial Processing Peptidase
D008666:Metalloendopeptidases; D011480:Protease Inhibitors; D011994:Recombinant Proteins; D015032:Zinc; D000096122:Mitochondrial Processing Peptidase