Structural requirements for the utilization of ascorbate analogues in the prolyl 4-hydroxylase reaction.
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.
Tschank
G
G
Hoechst AG, Frankfurt/Main, Federal Republic of Germany.
Sanders
J
J
Baringhaus
K H
KH
Dallacker
F
F
Kivirikko
K I
KI
Günzler
V
V
D000595:Amino Acid Sequence; D000818:Animals; D001205:Ascorbic Acid / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism*; D002642:Chick Embryo; D008861:Microsomes / Q000201:enzymology; D008969:Molecular Sequence Data; D015394:Molecular Structure; D011393:Procollagen-Proline Dioxygenase / Q000302:isolation & purification / Q000378:metabolism*
D011393:Procollagen-Proline Dioxygenase; D001205:Ascorbic Acid