Biomedical subjects

P Luciano

Publications and source records attributed to P Luciano.

3 recordsLinked to original sources

PubMed1998

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.

Escherichia coli↗

PubMed1997

Functional cooperation of the mitochondrial processing peptidase subunits.

Domains important for the activity of the heterodimeric mitochondrial processing peptidase (MPP) were investigated, by inserting one alanine residue at ten positions along the polypeptide chain of the beta-subunit (beta-MPP). An alanine residue inserted after Glu70, Ser114, Lys215 and Ser314 respectively, abolished the cleavage activity of MPP. When the alpha-subunit (alpha-MPP) was co-expressed with N-terminal hexa-histidine tagged beta-MPP, alpha-MPP was co-eluted from a nickel-derivatized affinity resin, with a 1:1 stochiometry, both with wild-type beta-MPP and with the mutants with alanine inserted after Ser114 and Ser314. The mutants with alanine inserted after Glu70 and Lys215 did not associate with alpha-MPP. The mutagenesis studies indicate that: (1) the whole HXXEHX76H region of beta-MPP is important for the proper conformation of the active site of MPP and may also be in contact with alpha-MPP; (2) the non-conserved central region surrounding Lys215 is involved in the interaction with alpha-MPP; and (3) the carboxy-terminal region of beta-MPP surrounding Ser314 is also of importance for the catalysis. Cross-linking studies indicated that purified alpha-MPP bound a precursor protein in the absence of any beta-MPP. Furthermore, the interaction of MPP and its subunits with a peptide substrate, as analyzed by surface plasmon resonance, showed that alpha-MPP bound a peptide substrate as efficiently as MPP. The data suggest that the alpha-subunit is responsible for the binding of mitochondrial presequences prior their presentation to the catalytic site of MPP.

Alanine↗

PubMed1996

The mitochondrial processing peptidase: function and specificity.

Targeting signals of mitochondrial precursors are cleaved in the matrix during or after import by the mitochondrial processing peptidase (MPP). This enzyme consists of two nonidentical alpha- and beta-subunits each of molecular weight of about 50 kDa. In mammals and fungi, MPP is soluble in the matrix, whereas in plants the enzyme is part of the cytochrome bc1 complex. MPP is a metalloendopeptidase which has been classified as a member of the pitrilysin family on the basis of the HXXEHX76E zinc-binding motif present in beta-MPP. Both subunits of MPP are required for processing activity. The alpha-subunit of MPP, which probably recognizes a three-dimensional motif adopted by the presequence, presents the presequence to beta-MPP, which carries the catalytic active site. MPP acts as an endoprotease on chemically synthesized peptides corresponding to mitochondrial presequences. Matrix-targeting signals and MPP cleavage signals seem to be distinct, although the two signals may overlap within a given presequence. The structural element helix-turn-helix, that cleavable presequences adopt in a membrane mimetic environment, may be required for processing but is not sufficient for proteolysis. Binding of the presequence by alpha-MPP tolerates a high degree of mutations of the presequence. alpha-MPP may present a degenerated cleavage site motif to beta-MPP in an accessible conformation for processing. The conformation of mitochondrial presequences bound to MPP remains largely unknown.

Kinetics↗