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Structural requirements for the utilization of ascorbate analogues in the prolyl 4-hydroxylase reaction.

ABSTRACT: The ability of structural analogues of ascorbate to serve as substitutes for this reducing agent in the prolyl 4-hydroxylase reaction was studied. In experiments using the purified enzyme, variations of the compounds' side chain were compatible with co-substrate activity. The presence of very large hydrophobic substituents or a positively charged group caused an increase in the observed Km values. A negative charge and smaller modifications did not change the affinity to the enzyme when compared with L-ascorbate. 6-Bromo-6-deoxy-L-ascorbate had a lower Km 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 Vmax. values with all compounds were similar. The reaction of microsomal prolyl 4-hydroxylase was supported by D-isoascorbate, O6-tosyl-L-ascorbate and 5-deoxy-L-ascorbate, giving the same dose-response behaviour as L-ascorbate itself. Again, 6-bromo-6-deoxy-L-ascorbate gave a lower Km and a similar Vmax. value. L-Ascorbic acid 6-carboxylate produced substrate inhibition at concentrations above 0.3 mM. The Km and Vmax. values calculated from concentrations up to 0.2 mM were similar to those of L-ascorbate. The enzyme activity observed with 6-amino-6-deoxy-L-ascorbate was very low in the microsomal hydroxylation system. The calculated Vmax. value was lower than that of L-ascorbate, suggesting a restriction of the access of this compound to the enzyme.

SUBMITTER: Tschank G

PROVIDER: S-EPMC1138125 | biostudies-other | 1994 May

REPOSITORIES: biostudies-other

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Structural Basis of Prolyl Hydroxylase Domain Inhibition by Molidustat.

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