COMPARISON OF CLEAVAGE SITE-SPECIFICITY OF GELATINASE-A AND GELATINASE-B USING COLLAGENOUS PEPTIDES

The gelatinases (type IV collagenases) are members of the matrix metal loproteinase family that not only have a high degree of structural hom ology but are known to be nearly identical in their digestion profile against macromolecular substrates. We have shown previously that the p referred cleavage sites in the hydrolysis of type I gelatin, catalyzed by gelatinase A (72 kDa type IV collagenase), are bracketed by hydrox yproline in the P5 and P5' positions. In this report, a kinetic invest igation using a series of collagenous dodecylpeptides in which the P5 and P5' hydroxyprolines were systematically varied and used as substra tes for recombinant human gelatinase A, we show that replacement with either proline or alanine always resulted in increased K-m. In contras t, substitution of the hydroxylated amino acids tyrosine and serine at P5 and P5' reduced the K, significantly, indicating that the hydroxyl moiety of the hydroxyproline is the functional group responsible for favorable enzyme-substrate affinity. This was shown by the k(cat)/K-m ratio, which was doubled by the substitution of serine in that site. C leavage of the same series of dodecylpeptides by recombinant human gel atinase B (92 kDa type TV collagenase) showed a very different kinetic profile for which no patterns were discernible. In subsequent compari sons of the two enzymes, it was found that gelatinase B cleaved the th iopeptolide substrate AcProLeuGly-S-LeuLeuGly-OC2H5 at double the velo city of gelatinase A. Ln contrast, gelatinase A digested type I gelati n about 2.5-times faster than gelatinase B. SDS-PAGE analysis of gelat in cleavage products showed different patterns of product peptides for each enzyme. Further comparisons of the proteinases using synthetic p eptide substrates with variations in size and in substituents at the P 2' site again showed marked kinetic differences. Although these two ma trix metalloproteinases seem similar in that they are both gelatinolyt ic and can degrade a nearly identical battery of macromolecular matrix components including type IV collagen, it is clear from these results that they are very different enzymatically. Since the regulatory port ions of gelatinases A and B differ markedly, it has been assumed that the enzymes serve the same function, but respond to different stimuli. The differences in substrate specificity described herein suggest tha t their proposed physiological roles may require reevaluation.