Mevalonate pyrophosphate decarboxylase catalyzes the decarboxylation of mevalonate pyrophosphate to isopentyl pyrophosphate. A study of the mechanism of inhibition by ...3-hydroxy-3-(fluoromethyl)-5-pyrophosphopentanoic acid (II) was undertaken. It was found that II is a competitive inhibitor (K$\sb{\rm i}$ = 0.01 $\mu$M) of the enzyme reaction (Reardon, J. E. & Abeles, R. H. (1987) Biochemistry 26, 4717-4722; Nave, J. F., d'Orchymont, H., Ducep, J. B., Piriou, F., & Jung, M. J. (1985) Biochem. J. 227, 247-254). We have now observed that II is decarboxylated 2500-fold more slowly than mevalonate pyrophosphate (3-hydroxy-3-methyl-5-pyrophosphopentanoic acid, I). The enzyme was exposed to saturating concentrations of II and ATP and then passed through a Penefsky column to remove excess substrate. The enzyme was denatured immediately upon emerging from the Penefsky column. Nearly 1 equivalent of both 3-phospho-3-(fluoromethyl)-5-pyrophosphopentanoic acid and ADP was bound to the enzyme. 3-Hydroxy-5-pyrophosphopentanoic acid (III) is phosphorylated at the secondary hydroxyl group and released from the enzyme without decarboxylation. This reaction is 30-fold slower than the rate of decarboxylation of I. The introduction of the 3-fluoromethyl group as well as the removal of the 3-methyl group tend to destabilize an alpha carbocation, therefore these compounds result in low rates of decarboxylation. Further support for a carbocationic transition state is provided by the finding that N-methyl-N-carboxymethyl-2-pyrophosphoethanolamine (IV) inhibits the enzyme reaction with K$\sb{\rm i}$ = 0.75 $\mu$M, but 3-methyl-5-pyrophosphopentanoic acid (v) does not. IV is probably a transition-state analog in which the positively charged nitrogen atom is analogous to the carbocation. Inhibitors of mevalonate pyrophosphate decarboxylase are likely to contain membrane-impermeable phosphate groups. We found that monophosphorylated inhibitors bind about 1000-fold less than their pyrophosphorylated forms. Compounds that have the pyrophosphate group of mevalonate pyrophosphate replaced with N-hydroxy-glycine bind poorly to mevalonate pyrophosphate decarboxylase.
The effect of acidic phospholipids on the activity of a Na(+)-dependent amino acid transporter (A system) from Ehrlich ascites cell plasma membranes was examined. Plasma membranes were solubilized in ...cholate/urea and reconstituted with Ba2(+)-precipitated asolectin (soybean phospholipid free of anionic phospholipids) replenished with different acidic phospholipids. In the absence of added acidic phospholipids, transport activity was very low. However, three acidic lipids cardiolipin greater than phosphatidic acid (PA) greater than phosphatidylinositol were capable of restoring transport activity (in the order given) to proteoliposomes made from Ba2(+)-precipitated asolectin, while other acidic phospholipids (phosphatidylserine and phosphatidylglycerol) were much less active in this respect. For restoration of optimal activity, PA containing at least one unsaturated fatty acyl moiety, particularly in the beta position, was required. PA containing only saturated fatty acids in the beta and gamma positions was largely inactive. No difference in restoration of function was observed on varying the saturated fatty acyl chain length in PA from 10 carbons to 18 carbons. The specific effects of PA on the A-system transporter were not shared by the Na(+)-independent amino acid exchange system (L system) or the glucose transport system. Treatment with poly(ethylene glycol) 8000 was shown to reduce the nonspecific permeability of the reconstituted proteoliposomes and to enhance Na(+)-dependent amino acid transport.
