ANALYSIS OF THE CONTRIBUTION OF THE HINGE REGION OF HUMAN NEUTROPHIL COLLAGENASE (HNC, MMP-8) TO STABILITY AND COLLAGENOLYTIC ACTIVITY BY ALANINE SCANNING MUTAGENESIS

Analysis of the hinge region of neutrophil collagenase by alanine scan ning mutagenesis revealed that this sequence motif has a pronounced ef fect on the stability and collagenolytic activity of the active enzyme , The mutagenesis of the amino acid residues in the P-1' position of t he two autoproteolytically cleaved peptide bonds (Leu(243) and Ile(248 )) to Ala showed that the mutant enzymes were more resistant to autopr oteolysis, However, these mutants were not completely stable and autop roteolysis occurred mainly at the Ala(239) - Ile(240) peptide bond and the half-life of the active enzyme was increased by 50%, In contrast, mutagenesis of Pro(247) --> Ala (P-1 of the minor cleavage site Pro(2 47) - Ile(248)) lead to increased susceptibility of the enzyme to auto proteolysis, However,when the other P-1 position Gly(242) was altered to Ala no effect on stability was observed, The analysis of the abilit y of the mutant active enzymes to hydrolyse C-14-type I collagen was a ssessed and our results demonstrate that the hinge sequence motif of n eutrophil collagenase is important for collagenolytic activity. The al teration of the Gly(242)-Leu-Ser-Ser-Asn-Pro-Ile-Gln-Pro(247) sequence motif to Gly(242)-Ala-Ala-Ala-Ala-Pro-Ala-Ala-Pro(247) showed that th e collagenolytic activity was reduced by 68.4%, In addition, mutagenes is of the downstream sequence motif Pro(247)-Thr-Gly-Pro-Ser-Thr-Pro-L ys-Pro(258) to Pro(247)-Ala-Ala-Pro-Ala-Ala-Pro-Ala-Pro(258) had an ev en more marked effect on the collagenolytic activity, which was reduce d by 87.4%, When the Pro residues in the hinge motif (Pro(247), Pro(25 0), Pro(253) and P-256) were altered to Ala the collagenolytic activit y dropped to 1.5% of the value observed for wild-type enzyme. (C) 1997 Federation of European Biochemical Societies.