THE KINETICS OF THE HYDROXYLATION OF PROCOLLAGEN BY PROLYL 4-HYDROXYLASE - PROPOSAL FOR A PROCESSIVE MECHANISM OF BINDING OF THE DIMERIC HYDROXYLATING ENZYME IN RELATION TO THE HIGH KCAT/KM RATIO AND A CONFORMATIONAL REQUIREMENT FOR HYDROXYLATION OF -X-PRO-GLY- SEQUENCES

Prolyl 4-hydroylase modifies only approx. 5% of the hydroxylatable prolyl residues in procollagen at a relatively high rate, after which the rate of further hydroxylation rapidly decreases. This suggests that the probability to exist in a defined hydroxylation-committed conformation differs between the numerous -X-Pro-Gly- sequences in the substrate. The enzyme reaction is characterized by the unusually high k(cat)/K(m) ratio of 3.10(9) M-1 S-1. To explain these kinetic features, an extremely high second-order rate constant for the association of enzyme and the subset of rapidly hydroxylated prolyl residues has to be assumed. A two-step mechanism is proposed in which diffusional constraints on the rate of association of prolyl 4-hydroxylase with hydroxylatable prolyl residues can be overcome. Upon encountering a random coil pro-alpha chain, the dimeric enzyme is first 'aspecifically' bound, followed by rapid transfers between different segments of the flexible peptide substrate via fast transitions between 'aspecific' single and double bound intermediate states. The rate of the second step, the productive (specific) binding of hydroxylation-committed -X-Pro-Gly- sequences to the active site, can be enhanced significantly by such an, in essence, 'one-dimensional' search. This processive mechanism of binding does not necessarily imply many hydroxylation reactions during one encounter between enzyme and a peptide with several substrate sites as suggested previously in a slightly different model (De Waal, A. and De Jong, L. (1988) Biochemistry 27, 150-155).