KINETICS OF DIPEPTIDYL PEPTIDASE-IV PROTEOLYSIS OF GROWTH HORMONE-RELEASING FACTOR AND ANALOGS

The kinetics and selectivity of proteolysis of synthetic human growth hormone-releasing factor and analogs by purified human placental dipeptidyl peptidase IV (DPP IV) were studied by HPLC. The initial rates of Ala2-Asp3 cleavage (pH 7.8, 37-degrees-C, S(o) = 0.15 mM) were all approx. 5-mu-mol min-1 mg-1 for the parent hormone, GRF(1-44)-NH2, and the fragments, GRF(1-29)-NH2 and GRF(1-20)-NH2. Lower activities observed for GRF(1-11)-OH, GRF(1-3)-OH, and cyclic lactam analogs indicate S1'-S(n)' binding. Assays of [Trp6]-GRF(1-29)-NH2 versus [D-Trp6]-GRF(1-29)-NH2 indicate an S4' binding cavity. Peptides with D-configuration at P2, P1 or P1' and desNH2Tyr1 and N-MeTyr1 analogs of GRF were not cleaved. Catalytic parameters for the P1-substituted analogs [X2,Ala15]-GRF(1-29)-NH2 were found to vary with X as follows, K(m): Abu < Ala < Pro < Val < Ser < Gly << Leu; k(cat): Pro > Ala > Abu > Ser > Gly >> Leu > Val; k(cat)/K(m): Abu > Pro > Ala >> Ser > Gly = Val >> Leu. K(m) is at a minimum and k(cat)/K(m) at a maximum, for a hydrophobic P1 side-chain of about 0.25 nm in length, i.e., the ethyl side-chain of alpha-aminobutyric acid (Abu) is very close to optimal. These results further define the S1 selectivity of DPP IV and may be useful in the design of DPP IV resistant GRF analogs that can be produced by recombinant DNA methods and the design of DPP IV inhibitors.