Pyruvate Dehydrogenase Complex from Higher Plant Mitochondria and Proplastids: Kinetics

Abstract

be competitive with the substrate for that enzyme unless the central complexes are kinetically important, in which case they will be noncompetitive. This prediction has been confirmed for acetyl-CoA and NADH in the bovine kidney and pig heart complexes (7, 16). Other product inhibition patterns should be uncompetitive except for the interaction of one product and the substrate of the subsequent enzyme when it will be noncompetitive, if the reaction at that site is random sequential. In the mammalian complexes (7, 16), acetyl-CoA and NADH are both uncompetitive against pyruvate but acetyl-CoA and NADH are noncompetitive against NAD+ and CoA, respectively. This may indicate that these products can act as dead end inhibitors, or the binding of products may sterically hinder the binding of substrates on the adjacent enzyme. A third possibility is that there may be protein-protein interactions between the second and third enzymes of the complex (7, 16).The steady-state kinetic analyses to determine the reaction mechanism of the plant complex have not been described. The regulatory properties of the enzyme from potato tubers have been described and some preliminary kinetics performed (5). Since some of the interactions between products and substrates are other than those predicted by theory, it is important that further kinetics be performed. Also, a steady-state kinetic analysis may be used to determine whether the mitochondrial enzyme and the enzyme from proplastids (11) are significantly different.The pyruvate dehydrogenase complex consists of three enzymes: pyruvate dehydrogenase (pyruvate lipoate oxidoreductase, EC 1.2.4.1), lipoate transacetylase (acetyl-CoA dihydrolipoate S-acetyl transferase, EC 2.3.1.12), and dihydrolipoamide dehydrogenase (NADH lipoamide oxidoreductase, EC 1.6.4.3).The reactions of these three enzymes are shown in the accompanying paper (11) which also gives an account of the purification and preliminary characteristics of the pea mitochondrial complex and isolation of the proplastid complex. The reactions of the complex occur at three distinct sites on the complex located on three different proteins. The nature of the reactions at each of these sites suggests that the over-all mechanism should be ping pong. A rate equation for a three-site ping-pong mechanism has been derived by Cleland (4) and from this it was predicted that the pyruvate dehydrogenase complex should show initial substrate velocity patterns which are parallel when plotted in double reciprocal form, regardless of which substrate is varied at fixed levels of a second. Such a pattern has been demonstrated for the pyruvate dehydrogenase complexes from bovine kidney (16) and pig heart (7). The products of each enzyme component should I

Some Kinetic and Regulatory Properties of the Pea Mitochondrial Pyruvate Dehydrogenase Complex

“…The values obtained were in the range of those previously reported for PDC from etiolated pea shoots (29) and mitochondrial PDCs from other plant sources (5,17,28). The K, value for NADH is substantially lower than the Km NAD suggesting that product inhibition is of regulatory significance.…”

“…It has been shown that like all mitochondrial PDCs thus far examined, the pea leaf complex is regulated in part by covalent modification (18), and the PDC from etiolated pea tissues exhibits product inhibition (29). While the pea mitochondrial PDC is the most thoroughly studied plant PDC, there are some inconsistencies in the reported results.…”

“…Most studies of the mitochondrial PDC from plant sources have dealt with the kinetic mechanism or with phosphorylation/ dephosphorylation and both of these aspects have been the subject of previous reports on pea mitochondrial PDC (18,29). There has, however, been little basic information available on the enzymic characteristics of the complex from green leaftissue.…”

“…A competitive inhibition pattern was observed when varying CoA concentrations in the presence of fixed varying acetyl-CoA, from bovine heart or kidney mitochondria and Ricinus endosperm plastids (29), as well as for Ricinus mitochondrial PDC (BJ Rapp, DD Randall, unpublished data) and pea chloroplast PDC (PJ Camp, DD Randall, unpublished data). The only exception is with pea mitochondrial PDC, where Thompson eX al.…”

“…We recently reviewed the available knowledge of plant PDCs (14). Most reports have concentrated upon the subcellular localization of the complex in plant tissues (9,21,24,25), the kinetic mechanism (26,29), and regulation of the mitochondrial complex by reversible phosphorylation (16-18, 20, 27).…”