FATTY-ACIDS AND ANIONIC PHOSPHOLIPIDS ALTER THE PALMITOYL COENZYME-A KINETICS OF HEPATIC MONOACYLGLYCEROL ACYLTRANSFERASE IN TRITON X-100 MIXED MICELLES
Ra. Coleman et al., FATTY-ACIDS AND ANIONIC PHOSPHOLIPIDS ALTER THE PALMITOYL COENZYME-A KINETICS OF HEPATIC MONOACYLGLYCEROL ACYLTRANSFERASE IN TRITON X-100 MIXED MICELLES, Biochemistry, 35(29), 1996, pp. 9576-9583
In order to gain a better understanding of the kinetics of activation
and inhibition of hepatic monoacylglycerol acyltransferase (MGAT) (EC
2.3.1.22) by fatty acid, we examined the effect of fatty acid with res
pect to MGAT's long-chain acyl-CoA substrate in Triton X-100 mixed mic
elles, At concentrations between 2.5 and 5.3 mol %, oleic acid stimula
ted MGAT activity 2-fold, whereas oleic acid inhibited MGAT at concent
rations higher than 7.5 mol %. The dependence on palmitoyl-CoA was hig
hly cooperative with a Hill constant of greater than 2.4. When present
at less than 3 mol %, oleic acid eliminated the lag in the dependence
curve. When concentrations of oleic acid were higher than 3 mol % Mic
haelis-Menton kinetics were observed with an apparent K-m value of abo
ut 54 mu M for palmitoyl-CoA but with progressively decreasing V-max v
alues, This effect was not observed with octanoic acid, suggesting tha
t the medium-chain fatty acid is unable to associate stably with the m
ixed micelle and, thus, cannot substantially alter substrate affinity.
When anionic phospholipids were tested, phosphatidic acid, lysophosph
atidic acid, phosphatidylserine, and phosphatidylinositol eliminated s
ome of the lag in activation by palmitoyl-CoA. At high molar concentra
tions of the anionic lipid activators, apparent K-m values ranged from
77 mu M for phosphatidic acid to 196 mu M for phosphatidylinositol. Z
witterionic phospholipids had no effect, nor did the non-phospholipid
activators bovine serum albumin or sn-1,2-diacylglycerol. CaCl2, but n
ot neomycin or KCl, could overcome the inhibitory effect of oleic acid
; thus, the inhibitory effect of fatty acid did not appear to occur by
electrostatic interactions, These blockers did not change the effects
observed with the anionic phospholipid activators or with the inhibit
or, sphingosine. An altered K-m for palmitoyl-CoA in the presence of f
atty acid or anionic phospholipid suggests that both long-chain fatty
acids and phospholipid cofactors may induce a conformational change in
MGAT, thereby altering the enzyme's affinity for its long-chain acyl-
CoA substrate, These data further support the hypothesis that tile syn
thesis of glycerolipids via the monoacylglycerol pathway may be highly
regulated via a variety of lipid second messengers such as phosphatid
ic acid and diacylglycerol, as well as by the influx of fatty acids de
rive hum high-fat diets, or from the hydrolysis of adipocyte triacylgl
ycerol during fasting or diabetes.