STRUCTURAL REQUIREMENTS FOR THE UTILIZATION OF ASCORBATE ANALOGS IN THE PROLYL 4-HYDROXYLASE REACTION

The ability of structural analogues of ascorbate to serve as substitut es for this reducing agent in the prolyl 4-hydroxylase reaction was st udied. In experiments using the purified enzyme, variations of the com pounds' side chain were compatible with co-substrate activity. The pre sence of very large hydrophobic substituents or a positively charged g roup caused an increase in the observed K-m values. A negative charge and smaller modifications did not change the affinity to the enzyme wh en compared with L-ascorbate. 6-Bromo-6-deoxy-L-ascorbate had a lower K-m than the physiological reductant. Substitution at the -OH group in ring position 3 prevented binding to the enzyme. The same pattern of activity was observed when the full and uncoupled prolyl 4-hydroxylase reactions were studied. The V-max. values with all compounds were sim ilar. The reaction of microsomal prolyl 4-hydroxylase was supported by D-isoascorbate, O-6-tosyl-L-ascorbate and 5-deoxy-L-ascorbate, giving the same dose-response behaviour as L-ascorbate itself. Again, 6-brom o-6-deoxy-L-ascorbate gave a lower K-m and a similar V-max. value. L-A scorbic acid 6-carboxylate produced substrate inhibition at concentrat ions above 0.3 mM. The K-m and V-max. values calculated from concentra tions up to 0.2 mM were similar to those of L-ascorbate. The enzyme ac tivity observed with 6-amino-6-deoxy-L-ascorbate was very low in the m icrosomal hydroxylation system. The calculated V-max. value was lower than that of L-ascorbate, suggesting a restriction of the access of th is compound to the enzyme.