Structure and function of microplasminogen: I. Methionine shuffling, chemical proteolysis, and proenzyme activation.
We have cloned and expressed microplasminogen (mPlg), consisting of the N-terminal undecapeptide of human glu-Plg spliced to its proenzyme domain. This truncated (approximately 28 kDa) proenzyme retained the distinctive catalytic activities of the larger parent. Replacement of M residues followed by M shuffling permitted subsequent scission by site-directed chemical proteolysis (in CNBr/formic acid) without impairing any of the protein's characteristic properties. Activation of chymotrypsinogen-related zymogens occurs by limited proteolysis; the newly liberated, highly conserved N-terminus (VVGG) forms a salt bridge with an aspartyl residue immediately upstream of the active site serine. The role of both of these elements in mPlg activation was probed using protein engineering and site-directed proteolysis to alter the length and amino acid composition of the N-terminus, and to replace the aspartate. All modifications affected both Km and Kcat. The results identify some structural parameters of the N-terminus required for proenzyme activation.
Department of Pharmacological Sciences, SUNY at Stony Brook 11794-8651, USA.
J
J
Wang
B
B
Brdar
E
E
Reich
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003488:Cyanogen Bromide / Q000737:chemistry*; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005561:Formates / Q000737:chemistry*; D006801:Humans; D008715:Methionine / Q000737:chemistry*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010958:Plasminogen / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering; D011994:Recombinant Proteins; D015183:Restriction Mapping
D004792:Enzyme Precursors; D005561:Formates; D011994:Recombinant Proteins; C030544:formic acid; D010958:Plasminogen; D008715:Methionine; D003488:Cyanogen Bromide