Time-dependent inactivation of chick-embryo prolyl 4-hydroxylase by coumalic acid. Evidence for a syncatalytic mechanism

Abstract

From the structure-activity relationships of known competitive inhibitors, coumalic acid (2-oxo-1,2H-pyran-5-carboxylic acid) was deduced to be a potential syncatalytic inhibitor for chick-embryo prolyl 4-hydroxylase. The compound caused time-dependent inactivation, the reaction rate being first-order. The inactivation constant was 0.094 min-1, the Ki 17 mm and the bimolecular rate constant 0.09 M-1 -s-1. Human prolyl 4-hydroxylase and chick embryo lysyl hydroxylase were also inactivated, though to a lesser extent. Inactivation could be prevented by adding high concentrations of 2-oxoglutarate or its competitive analogues to the reaction mixture. In Lineweaver-Burk kinetics, coumalic acid displayed S-parabolic competitive inhibition with respect to 2-oxoglutarate. The inactivation reaction had cofactor requirements similar to those for the decarboxylation of 2-oxoglutarate. Enzymic activity was partially preserved in the absence of iron, but the rescue was incomplete, owing to decreased stability of the enzyme under this condition. Coumalic acid also decreased the electrophoretic mobility of the a-subunit, but the f-subunit was not affected. Prolonged incubation of coumalic acid above pH 6.8 led to loss of its inactivating potency, owing to hydrolysis. It is concluded that the inactivation of prolyl 4-hydroxylase by coumalic acid is due to a syncatalytic mechanism. The data also suggest that the 2-oxoglutarate-binding site of the enzyme is located within the a-subunit.

The HAG Mechanism: A Molecular Rationale For The Therapeutic Application Of Iron Chelators In Human Diseases Involving the 2-Oxoacid Utilizing Dioxygenases

“…Thus, if the lysine residue which forms the wild-type subsite I required for salt bridge formation, is mutated to an arginine moiety, the size of that subsite within the confined space of the pocket increases by approximately 13 Å 3 , and consequently, hinders the binding of substrates and inhibitors that ionically interact with it, decreasing their affinity by up to one order of magnitude [64]. If they are small enough to fit into the pocket, nonpeptide syncatalytic inhibitors label only the catalytic subunits of prolyl 4-hydroxylase, as shown for coumalic acid (calculated surface area 131 Å 2 , calculated volume 110 Å 3 ) [23]. By contrast, much larger syncatalytic inhibitors, e.g.…”

“…2,4-PDCA suppressed the biosynthesis of only collagen and collectin, and did not affect protein biosynthesis in general [21]. Several sets of competitive and of enzyme-activated irreversible inhibitors were soon discerned among existing chemicals [22][23][24][25][26]. Arranged into analog series [20,22,27], they were used to map the geometry of the active site pocket, to probe the apoenzymeimposed steric control of catalytic tripod assembly, and to test the first stage of the ligand reaction, i.e.…”

Selective inhibition of prolyl 4-hydroxylases by bipyridinedicarboxylates

“…18 Compounds of this nature typically display values of IC 50 or K i that are less than the value of K m for AKG (which is 20 μM for human CP4H1). 19 There are also examples of electrophilic AKG mimics such as coumalic acid 20 and 4-oxo-5,6-epoxyhexanoic acid 21 that appear to inhibit CP4H via covalent modification of the active site. Compounds of this nature could have intriguing chemical and biological utility.…”

Syncatalytic inactivation of prolyl 4-hydroxylase by anthracyclines

“…Great progress has been made in designing inhibitory competitive analogues of 2-oxoglutarate and ascorbate (Hanauske-Abel, 1983;Majamaa et al, 1984Majamaa et al, , 1986, and coumalic acid has recently been characterized as the first substance that appears to inactivate prolyl 4-hydroxylase by a syncatalytic mechanism, i.e. enzyme-activated irreversible inhibitor, acting as a 2-oxoglutarate analogue (Giinzler et al, 1987). The class I anthracyclines doxorubicin and daunorubicin ( Fig.…”