Glutamate residues required for substrate binding and cleavage activity in mitochondrial processing peptidase.
Mitochondrial processing peptidase, a metalloendopeptidase consisting of alpha- and beta-subunits, specifically recognizes a large variety of mitochondrial precursor proteins and cleaves off N-terminal extension peptides. The enzyme requires the basic amino acid residues in the extension peptides for effective and specific cleavage. To elucidate the mechanism involved in the molecular recognition of substrate by the enzyme, several glutamates around the active site of the rat beta-subunit, which has a putative metal-binding motif, H56XXEH60, were mutated to alanines or aspartates, and effects on kinetic parameters, metal binding, and substrate binding of the enzyme were analyzed. None of mutant proteins analyzed was impaired in dimer formation with the alpha-subunit. Mutation of glutamates at positions 79, 129, and 136, in addition to an active-site glutamate at position 59, resulted in a marked decrease in cleavage efficiency. Together with sequence alignment data, glutamate 136 appears to be involved in metal binding. Glutamate 129 is mostly responsible for the catalysis, as there was a considerable decrease in kcat value by the mutation. Mutation of glutamate 79 led to decrease in kcat value and increase in Km values. Substrate binding experiments using an environmentally sensitive fluorescence probe attached to the peptide showed that the mutation caused a remarkable environmental change at the binding site to the N-terminal region of the substrate peptide and decreased binding of the peptide, thereby suggesting that glutamate 79 participates primarily in substrate binding. Thus, some glutamate residues required for substrate binding and cleavage activity have been identified.
Kitada
S
S
Department of Chemistry, Faculty of Science, Kyushu University, Fukuoka 812-8581, Japan.
Kojima
K
K
Shimokata
K
K
Ogishima
T
T
Ito
A
A
D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis; D004926:Escherichia coli / Q000235:genetics; D018698:Glutamic Acid / Q000378:metabolism*; D006868:Hydrolysis; D008666:Metalloendopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D051381:Rats; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D000096122:Mitochondrial Processing Peptidase
D018698:Glutamic Acid; D008666:Metalloendopeptidases; D000096122:Mitochondrial Processing Peptidase