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. 1995 Sep;4(9):1758–1767. doi: 10.1002/pro.5560040911

Structure and function of microplasminogen: I. Methionine shuffling, chemical proteolysis, and proenzyme activation.

J Wang 1, B Brdar 1, E Reich 1
PMCID: PMC2143206  PMID: 8528074

Abstract

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.

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Selected References

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