The long term objective of this proposal is to establish the mechanism of interaction of aldehyde and ketone inhibitors developed in this laboratory with their respective metalloprotease targets. The inhibitors are all related to substrates but with the amide bond replaced with a ketomethylene group or a formylmethylene group. The target proteases of this proposal are: carboxypeptidase A, a model metalloprotease whose x-ray crystallographic structure is known; angiotensin converting enzyme, inhibitors of which are antihypertensive drugs in humans; and C. histolyticum collagenase, a model for mammalian collagenases which are thought to be important in the pathology of tumor metastasis, angiogenesis, and rheumatoid arthritis. An understanding of the details of interaction of carbonyl-containing inhibitors with these target enzymes should allow the design of inhibitors of other medically significant metalloproteases such as the mammalian collagenases and proteoglycanases. The hypothesis of this proposal is two-fold. First, all carbonyl-containing inhibitors are proposed to be capable of binding to their target metalloproteases accompanied by enzyme catalyzed addition of a water molecule to their carbonyl group. Second, the most powerful inhibitors are proposed to bind as transition intermediate analogs in which the tetrahedral water-inhibitor adduct is trapped in this configuration at the active site of the enzyme. This adduct presumably mimics a tetrahedral intermediate which occurs during normal substrate hydrolysis and is thus bound very tightly by the enzyme. The specific aims of this proposal are to identify which inhibitors are tetrahedrally hybridized when bound to the enzyme, to identify the nucleophile as water or not, and to establish whether the enzyme catalyzes the addition of water to the inhibitors. Proof that the most powerful inhibitors bind as tetrahedral adducts will be by 13C-nmr of enzyme complexed with inhibitor labeled with 13C in the carbonyl carbon atom. Proof that water is the nucleophile in the adduct will be by observing the isotopic upfield shift of the carbonyl carbon atom in H2-18-0, 2H2-16-0 and 2H2-18-0 as solvent. Determination of enzyme catalyzed oxygen exchange will be by mass spectrometry of 16-0-labeled inhibitors incubated with enzyme in 18-0-water or by 13C-nmr using inhibitor labeled with 16-0 and 13C in the carbonyl group incubated with enzyme in 18-0 water.
NIH ExPorter
NIH ExPorter