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

Abstract

We have cloned and expressed microplasminogen (mPlg), consisting of the N‐terminal undecapeptide of human glu‐P/g spliced to its proenzyme domain. This truncated (∼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. Copyright © 1995 The Protein Society