The canonical methionine 392 of matrix metalloproteinase 2 (gelatinase A) is not required for catalytic efficiency or structural integrity: probing the role of the methionine-turn in the metzincin metalloprotease superfamily.
Matrix metalloproteinases (MMPs) are an important family of extracellular proteases that process a variety of biologically significant molecules. MMPs are members of the metzincin superfamily of >770 zinc endopeptidases, which includes astacins, serralysins, adamalysins, leishmanolysins, and snapalysins. Metzincins are characterized by an absolutely conserved methionine residue COOH-terminal to the third histidine in the consensus sequence HEXXHXXGXX(H/D), where the histidine residues chelate a catalytic zinc ion. The canonical methionine is part of a tight 1,4-beta-turn that loops the polypeptide chain beneath the catalytic zinc ion, forming a hydrophobic floor to the Zn(2+) ion binding site. The role of this methionine is uncertain, but its absolute conservation indicates an essential catalytic or structural function. To investigate this hypothesis, we replaced Met-392 that forms the Met-turn of human MMP-2 (gelatinase A) by site-directed mutagenesis. The catalytic competence of leucine and serine mutants was assessed. (M392L)MMP-2 and (M392S)MMP-2 cleaved the physiological substrates gelatin, native type I collagen, and the chemokine monocyte chemoattractant protein-3 with similar efficiency to wild-type MMP-2. These mutants also cleaved two quenched fluorescent peptide substrates with a k(cat)/K(m) comparable to wild-type MMP-2 and underwent 4-aminophenylmercuric acetate-induced autoactivation with similar kinetics. (M392L)MMP-2 and (M392S)MMP-2 were inhibited by tissue inhibitor of metalloproteinases (TIMP)-1, -2, and -4 and by the zinc chelators 1,10-phenanthroline and a synthetic hydroxamate inhibitor, Batimastat, similar to the wild-type protein, indicating an unaltered active site topography. A tryptic susceptibility assay also suggested that (M392L)MMP-2 and (M392S)MMP-2 were correctly folded. These results challenge the dogma that this methionine residue and the Met-turn, which are absolutely conserved in all of the subfamilies of the metzincins, play an essential role in catalysis or active site structure.
Butler
Georgina S
GS
Departments of Oral Biological & Medical Sciences and Biochemistry & Molecular Biology, Center for Blood Research, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada.
Tam
Eric M
EM
Overall
Christopher M
CM
D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D002384:Catalysis; D003094:Collagen / Q000737:chemistry; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D004492:Edetic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006639:Histidine / Q000737:chemistry; D006801:Humans; D007477:Ions; D007700:Kinetics; D007930:Leucine / Q000737:chemistry; D020778:Matrix Metalloproteinase 2 / Q000737:chemistry* / Q000378:metabolism; D008715:Methionine / Q000737:chemistry*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009841:Oligonucleotides / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D010662:Phenylmercuric Acetate / Q000031:analogs & derivatives* / Q000737:chemistry; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012694:Serine / Q000737:chemistry; D013696:Temperature; D013997:Time Factors; D014357:Trypsin / Q000494:pharmacology; D015032:Zinc / Q000737:chemistry
D007477:Ions; D009841:Oligonucleotides; D010455:Peptides; D012694:Serine; D006639:Histidine; C010024:4-aminophenylmercuriacetate; D003094:Collagen; D004492:Edetic Acid; D008715:Methionine; D014357:Trypsin; D020778:Matrix Metalloproteinase 2; D007930:Leucine; D015032:Zinc; D010662:Phenylmercuric Acetate