Role of metal ions in coupling between chemical catalysis and conformational changes in ATP synthase.

Isolated chloroplast ATP synthase (CF0F1) was used for determination of the structure-function relation by measuring the effect of divalent metal ions on the properties of ATPase. Mg2+ ions were more efficient catalysts than Ca2+ ions as indicated by Kcat/Km of 55.2 and 5.4, respectively. Other activity parameters related to binding, such as the Km of MATP and Ki of MADP, indicated a stronger binding in the presence of Mg2+ as seen from a Mg2+/Ca2+ ratio of 2.8 and 3.8, respectively. Strong binding of Ca2+ ions with a Kd of 0.03 +/- 00.6 microM-1 was detected only in the presence of ADP probably because of the positive interactive effect of CaADP as indicated in the inhibition properties. Mg2+ ions were more efficient catalysts also in other forms of the enzyme such as in the thylakoid membrane, in isolated CF0F1 and in CF1. The Mg2+/Ca2+ ratio of Kcat/Km was 5.3, 10.2 and 1.5 for the thylakoid membrane enzyme, the isolated CF0F1 and the soluble CF1 respectively. This indicated that Ca2+ ions became less efficient catalysts in the more intact and integrated enzyme while Mg2+ ions were as efficient in all forms of the enzyme. Unlike Mg2+, Ca2+ ions also did not support proton-coupled ATP synthesis and ATP driven proton pumping. It is suggested that the differences in the ligand structure of these two ions might be the reason for the differential function. An average 0.3 A shorter bond length of octahedral first coordination in Ca2+ ions caused a weaker binding of CaATP than that of MgATP. The effect of differential binding is discussed in relation to the binding of the transition state intermediate and to the rate of product release.

Vanadate, a transition state inhibitor of chloroplast CF1-ATPase.

The activity of CF1-ATPase was inhibited by vanadate in an allosteric manner with respect to CaATP as substrate. The cooperative interaction was enhanced by preincubation of the enzyme in the presence of ADP and Ca2+ ions and of free divalent metal ions during assay of the activity. The strongest cooperative interaction with a Hill coefficient of 5.3 +/- 0.1 was found when the reaction was stopped after 30 s, before steady state was reached. Under these conditions, the concentration of an exchangeable ADP, tightly bound to one of the active sites on the enzyme, was shown to be the highest. A Ki of 12.4 +/- 1.2 microM for vanadate inhibition was determined under these conditions. Direct measurements with the aid of 51V NMR indicated that vanadate binds to CF1 in the presence of Ca2+ and ADP in a positive cooperative manner with a Hill coefficient of 2.3 +/- 0.2 and an average Kd of 0.3 +/- 0.04 nM. It was suggested that a formation of pentacovalent vanadyl-ADP at the active site caused the inhibition. Vanadyl-ADP was suggested to be a strong inhibitor, being an analogue of a pentacovalent phosphoryl-ADP, which is proposed to be the transition state intermediate of CF1.

Studies of the catalytic mechanism of microsomal UDP-glucuronyltransferase. Alpha-glucuronidase activity and its stimulation by phospholipids.

The rate at which a specific, purified form of microsomal UDP-glucuronyltransferase (designated as the GT2P type of this enzyme) catalyzes the hydrolysis of UDP-glucuronic acid was measured with pure, delipidated enzyme and enzyme reconstituted with different lysophosphatidylcholines. This activity of the GT2P type of UDP-glucuronyltransferase is referred to as alpha-glucuronidase activity. For delipidated enzyme, the rate of hydrolysis of UDP-glucuronic acid catalyzed by GT2P extrapolated to infinite concentrations of UDP-glucuronic acid was 1 X 10(-9) mol/min/mg of protein. This compares with a rate of glucuronidation of p-nitrophenol of 96 X 10(-9) mol/min/mg of enzyme, for delipidated enzyme. Addition of oleoyl- or myristoyllysophosphatidylcholine to GT2P did not affect the alpha-glucuronidase activity significantly. This activity was stimulated, however, in the presence of compounds that bind at the aglycone site but that do not undergo glucuronidation. alpha-Glucuronidase activity extrapolated to infinite concentration of UDP-glucuronic acid was 4.0 X 10(-9) mol/min/mg for delipidated enzyme assayed in the presence of less than saturating concentrations of p-nitrophenyl phenyl ether. Moreover, when the aglycone site of GT2P was occupied by ethers, the alpha-glucuronidase activity of this enzyme was enhanced by addition of phospholipids to delipidated enzyme. The extent of activation of the alpha-glucuronidase activity of GT2P, when the aglycone site was occupied, depended on the acyl chain of the lipid added to delipidated enzyme. These data indicate that the GT2P form of UDP-glucuronyltransferase catalyzes the hydrolysis of UDP-glucuronic acid at a significant rate and that lysophosphatidylcholines can influence this rate.

Evidence that UDP-glucuronyltransferase in liver microsomes at 37 degrees C is in a gel phase lipid environment.

UDP-glucuronyltransferase (EC 2.4.1.17) in intact, untreated microsomes from pig liver is activated by relatively low concentrations of UDP-N-acetylglucosamine. This property is absent after treatment of microsomes with detergents or phospholipases, and also is not a characteristic of pure, delipidated enzyme. Sensitivity to activation by UDP-N-acetylglucosamine was reconstituted, however, by incorporation of pure, delipidated enzyme into unilamellar bilayers of phosphatidylcholine that were in a gel phase. Warming of these bilayers to just above the temperature for the gel to liquid crystalline phase transition led to an abrupt loss of sensitivity of UDP-glucuronyltransferase to activation by UDP-N-acetylglucosamine. These experiments establish that sensitivity to activation by UDP-N-acetylglucosamine is an inherent property of UDP-glucuronyltransferase. The data also suggest that the lipid environment of UDP-glucuronyltransferase in intact, untreated microsomes can modulate the sensitivity of this enzyme to allosteric activation by UDP-N-acetylglucosamine, and that this lipid environment is in a gel phase in intact microsomes at 37 degrees C.

Evidence for an active site arginine in UDP-glucuronyltransferase.

2,3-Butanedione inactivates the pure form of UDP-glucuronyltransferase used in these experiments (GT2P) (EC 2.4.1.17) purified from pig liver microsomes. The kinetics of the reaction indicates that 2,3-butanedione reacts with two amino acids that affect activity. A rapid, partial inactivation is followed by a slower rate of inactivation that leads eventually to completely inactive enzyme. UDP-glucuronic acid and glucuronic acid, as compared with UDP, are effective as protectors against the slow, secondary phase of inactivation; no ligand tested protected against the rapid phase of inactivation. The lipid environment of GT2P was a determinant of the pseudo-first order rate constant for the slow phase of inactivation, but did not affect the rate of the rapid phase of inactivation. The data suggest that GT2P contains an active site arginine that interacts with the -COO- at C-6 of the glucuronic acid moiety of UDP-glucuronic acid.

Modulation of the number of ligand binding sites of UDP-glucuronyltransferase by the gel to liquid-crystal phase transition of phosphatidylcholines.

The kinetics of a pure, delipidated form of microsomal UDP-glucuronyltransferase is non-Michaelis-Menten when the enzyme is reconstituted into unilamellar vesicles of phosphatidylcholine that are in a gel phase. Double reciprocal plots of velocity as a function of the concentration of UDP-glucuronic acid show a downward curvature under these conditions. Binding studies indicate that the basis for the kinetic pattern is the presence of one high affinity and one low affinity binding site for UDP-glucuronic acid. The two classes of binding sites seem to be generated by the presence of two subunits that bind UDP-glucuronic acid within a single molecule of UDP-glucuronyltransferase. Melting the phospholipids from the gel phase to the liquid-crystal phase is associated with a switch from non-Michaelis-Menten to Michaelis-Menten kinetics for UDP-glucuronyltransferase. Binding studies for interaction of UDP-glucuronic acid with enzyme present in a liquid-crystal lipid phase indicate that the two binding sites for UDP-glucuronic acid do not become identical in this setting. Instead, one of the sites becomes nonfunctional. Binding studies carried out with UDP as ligand lead to similar results. There is a high affinity and a low affinity site for UDP when enzyme is reconstituted into a phospholipid bilayer in a gel phase. There is only one UDP binding site per holoenzyme when enzyme is reconstituted into a phospholipid bilayer in a liquid-crystal phase. Delipidated enzyme or enzyme reconstituted with lysophosphatidylcholine displays Michaelis-Menten kinetics. Binding studies show that these forms have only one binding site for UDP-glucuronic acid per holoenzyme. However, they have two nonidentical binding sites for binding of UDP. Thus, the physical properties of its phospholipid milieu influence the number of functional binding sites of UDP-glucuronyltransferase.

A comparison of the kinetic properties of two different forms of microsomal UDPglucuronyltransferase.

Two forms of UDPglucuronyltransferase (EC 2.4.1.17) have been purified from microsomes of pig liver. One form is free of phospholipids and the other contains a small amount of residual phospholipids. Each form, however, is responsive to activation on addition of purified phospholipids. Comparison of kinetic properties of these enzymes, after reconstitution with identical phospholipid environments, indicate that these are unique functional forms of UDPglucuronyltransferase. The two differ by as much as 100-fold in their rates of conjugation at Vm of p-nitrophenol. Relative rates of glucuronidation of a variety of phenolic aglycones are different for the two enzymes, which suggests different reaction mechanisms. The energetic basis for binding of UDP-glucuronic acid to the active sites is different for the two forms of UDPglucuronyltransferase. Moreover, one form, but not the other, binds Mn2+, which leads to modulation of kinetic properties.

Modulation by bicarbonate, phosphate, and maleate of the kinetics of adenosinetriphosphatase activity and of the binding of manganese ions to chloroplast coupling factor 1.

Bicarbonate, maleate, and phosphate were shown to modulate adenosinetriphosphatase (ATPase) activity in coupling factor 1 from chloroplasts. Kinetic analysis of the changes in the ratio between the apparent Km with and without effectors indicated that the stimulation of the activity by bicarbonate was a result of a decrease in the Km for MnATP2-. The inhibition by phosphate resulted from a decrease in the Ki for free ATP as a competitive inhibitor at pH 8. THe effectors did not change Vmax at this pH. However, at pH 6.5, both Km and Vmax of ATPase activity with MnATP2- were changed by maleate, yet the mode of inhibition by free ATP remained unaltered. In addition to decreasing the Km, bicarbonate induced a 10-fold decrease in the Kd for binding of Mn2+ at the two tight binding sites in the presence of ATP at pH 8. At pH 6.5, maleate also decreased both the Km for MnATP2- and the Kd for Mn2+ binding. A decrease in the Km of a substrate induced by an effector is likely to be a result of a decrease in the binding constant of the substrate. Therefore, these results are in harmony with the suggested assignment of the two tight binding sites of Mn2+ at the active sites of the enzyme.

A kinetic mechanism for modulation of the activity of microsomal UDP-glucuronyltransferase by phospholipids. Effects of lysophosphatidylcholines.

The affinity of delipidated microsomal UDP-glucuronyltransferase (EC 2.3.1.17) for UDP is greater than that for UDP-glucuronic acid. Measurement of KIglucuronic acid reveals that glucuronic acid binds to the enzyme. Hence, the difference in affinity of the enzyme for UDP versus UDP-glucuronic acid indicates that inherent binding energy for interactions between enzyme and this substrate is used for purposes other than enhancing binding. A reasonable interpretation of these data is that the binding of UDP-glucuronic acid to enzyme requires distortion of the substrate and/or the enzyme. Inherent binding energy due to interactions between enzyme and UDP and glucuronic acid is utilized to effect such distortions. This type of mechanism can cause significant rate enhancement. Phospholipid activators of UDP-glucuronyltransferase activate by amplifying this basic mechanism. Thus, addition of various species of lysophosphatidylcholine to the delipidated enzyme increase the activity at Vmax and enhance the affinity for UDP, glucuronic acid, and UDP-glucuronic acid. However, activators enhance the affinity of the enzyme for UDP-glucuronic acid to a significantly smaller extent than they enhance affinity for the UDP and glucuronic acid portions of the substrate. Calculations of the amount of binding energy for interactions between enzyme and UDP-glucuronic acid that can be used for stimulating activities at Vmax yield values in agreement with the observed enhancement of activities at Vmax for enzyme reconstituted with various types of lysophosphatidylcholine.