Paul A. Srere

[1] Citrate synthase. [EC 4.1.3.7. Citrate oxaloacetate-lyase (CoA-acetylating)]

Publisher Summary This chapter is dedicated to describing citrate synthase. The assay of citrate synthase is performed by coupling it to the transacetylase reaction. The disappearance of acetyl phosphate is followed by a hydroxamate method and the formation of citrate by the pentabromoacetone method. The malate dehydrogenase catalyzed reaction is used to generate the oxaloacetate for the citrate synthase reaction. Another method for assaying citrate synthase uses 14 C-acetyl-CoA and measures its incorporation in 14 C-citrate, which is isolated as a silver salt. Citrate synthase can be followed by measuring the appearance of the free SH group of the released CoASH; three such methods are discussed in the chapter. One method is to measure the oxidation of the CoASH by dichlorophenol- indophenol, which is accompanied by a decrease in absorbancy at 578 mμ. Another method measures the CoASH polarographically. The third method measures SH by the use of 5, 5’-dithiobis-(2-nitrobenzoate) (DTNB) (Ellmans reagent).

Macromolecular compartmentation and channeling.

One of the accepted characterizations of the living state is that it is complex to an extraordinary degree. Since our current understanding of the living condition is minimal and fragmentary, it is not surprising that our first descriptions are simplistic. However, in certain areas of metabolism, especially those that have been amenable to experimentation for the longest period of time, the simplistic explanations have been the most difficult to revise. For example, current texts of general biochemistry still view metabolism as occurring by a series of independent enzymes dispersed in a uniform aqueous environment. This notion has been shown to be deeply flawed by both experimental and theoretical considerations. Thus, there is ample evidence that, in many metabolic pathways, specific interactions between sequential enzymes occur as static and/or dynamic complexes. In addition, reversible interactions of enzymes with structural proteins and membranes is a common occurrence. The interactions of enzymes give rise to a higher level of complexity that must be accounted for when one wishes to understand the regulation of metabolism. One of the phenomena that occurs because of sequential enzyme interactions is the process of channeling. This article discusses enzyme interactions and channeling and summarizes experimental and theoretical results from a few well-studied examples.

The infrastructure of the mitochondrial matrix

Abstract The concentration of the enzymes in the matrix of rat-liver mitochondria is over 50% by weight and they probably exist and behave as a multienzyme complex rather than as enzymes in solution. Changes in the volume of the matrix causing alterations in the concentration of the enzymes and their substrates and metabolites probably play a role in metabolic control.

Macromolecular interactions: tracing the roots.

. Twomajor theses of that paper are first, thatthe importance of macromolecularinteractions is not ‘new’to biochemistry;and second, that contemporary scientistshave the responsibility to recognizeprevious contributions from otherscientists and integrate these findingswhenever possible into the new workbeing presented. The recent contributionto ‘Talking Points’by Kisters-Woike

Studies on cell adhesion. II. Adhesion of cells to surfaces of diverse chemical composition and inhibition of adhesion by sulfhydryl binding reagents

Abstract Rat hepatoma cells are able to adhere to many surfaces of diverse chemical compositions. Adhesion to different kinds of surfaces was inhibited when cells were treated with sulfhydryl binding reagents, but not with inhibitors of transport, respiration, and glycolysis. Inhibition of adhesion by p -mercuribenozate and arsenite was reversible; inhibition by HgCl 2 , iodoacetate and N -ethylmaleimide was not reversible. Cell adhesion is discussed in terms of current theories of chemical adhesion.

Enzyme-enzyme interactions and their metabolic role

There are continuing reports on the existence of complexes of sequential metabolic enzymes. New techniques for their detection have been described and include affinity electrophoresis and the use of anti‐idiotypic antibodies. Channeling of substrates has been reported for several systems as well as direct substrate transfer through dynamic enzyme associations. Kinetic parameters of metabolic control of organized systems have been formulated and tested in several systems. These recent results are expanding our understanding of metabolic processes and their control.

The Citrate Enzymes: Their Structures, Mechanisms, and Biological Functions

Publisher Summary The enzymes that catalyze lyase reactions on citrate to yield a C 2 and a C 4 unit are: citrate lyase, citrate synthase, and adenosine triphosphate (ATP) citrate lyase. Citrate lyase has been reported only in certain bacteria, citrate synthase has been found in all cells examined for it, and ATP citrate lyase has been found only in eukaryotic cells. The reaction catalyzed in common by these three enzymes is an aldol type, that is the reversible formation of a C—H bond from a C—C bond. It is possible that there exist some catalytic similarities in the three enzymatic reactions so that comparison of the results obtained with each enzyme might be useful in understanding the others. The citrate enzymes comprise a unique biological system in which three enzymes catalyze the same bond-breaking-making reaction. The fact that the simplest of these reactions, that catalyzed by citrate lyase, occurs in the most primitive organisms and that the most complex, the ATP citrate lyase reaction, occurs in the most recently evolved organisms, suggests a possible evolutionary relation between these enzymes.

Interaction between citrate synthase and mitochondrial malate dehydrogenase in the presence of polyethylene glycol

Abstract Pig heart citrate synthase and mitochondrial malate dehydrogenase interact in polyethylene glycol solutions as indicated by increased solution turbidity. A large percentage of both enzymes sediments when mixtures of the two in polyethylene glycol are centrifuged, whereas little if any of either enzyme sediments in the absence of the other. The observed interaction is highly specific in that neither cytosolic malate dehydrogenase nor nine other proteins showed evidence of specific interaction with either pig heart citrate synthase or mitochondrial malate dehydrogenase. Escherichia coli citrate synthase did not interact with pig heart citrate synthase, but did show evidence of interaction with pig heart mitochondrial malate dehydrogenase. The relation between enzyme behavior in polyethylene glycol solution and in the mitochondrion and the significance of possible in vivo interactions between citrate synthase and mitochondrial malate dehydrogenase are discussed.

Interaction between Citrate Synthase and Malate Dehydrogenase SUBSTRATE CHANNELING OF OXALOACETATE

The interactions between pig heart citrate synthase and mitochondrial malate dehydrogenase or cytosolic malate dehydrogenase were studied using the frontal analysis method of gel filtration and by precipitation in polyethylene glycol. This method showed that an interaction between citrate synthase and mitochondrial malate dehydrogenase occurred but no interaction between citrate synthase and cytosolic malate dehydrogenase. Channeling of oxaloacetate in the malate dehydrogenase and citrate synthase-coupled systems was tested using polyethylene glycol precipitates of citrate synthase and mitochondrial malate dehydrogenase, and citrate synthase and cytosolic malate dehydrogenase. The effectiveness of large amounts of aspartate aminotransferase and oxaloacetate decarboxylase, as competing enzymes for the intermediate oxaloacetate, was examined. Aspartate aminotransferase and oxaloacetate decarboxylase were less effective competitors for oxaloacetate when precipitated citrate synthase and mitochondrial malate dehydrogenase in polyethylene glycol was used at low ionic strength compared with free enzymes in the absence of polyethylene glycol or with a co-precipitate of citrate synthase and cytosolic malate dehydrogenase. Substrate channeling of oxaloacetate with citrate synthase-mitochondrial malate dehydrogenase precipitate was inefficient at high ionic strength. These effects could be explained through electrostatic interactions of mitochondrial but not cytosolic malate dehydrogenase with citrate synthase.

Adenosine triphosphate citrate lyase mediates hypocitraturia in rats.

Chronic metabolic acidosis increases proximal tubular citrate uptake and metabolism. The present study addressed the effect of chronic metabolic acidosis on a cytosolic enzyme of citrate metabolism, ATP citrate lyase. Chronic metabolic acidosis caused hypocitraturia in rats and increased renal cortical ATP citrate lyase activity by 67% after 7 d. Renal cortical ATP citrate lyase protein abundance increased by 29% after 3 d and by 141% after 7 d of acid diet. No significant change in mRNA abundance could be detected. Hypokalemia, which causes only intracellular acidosis, caused hypocitraturia and increased renal cortical ATP citrate lyase activity by 28%. Conversely, the hypercitraturia of chronic alkali feeding was associated with no change in ATP citrate lyase activity. Inhibition of ATP citrate lyase with the competitive inhibitor, 4S-hydroxycitrate, significantly abated hypocitraturia and increased urinary citrate excretion fourfold in chronic metabolic acidosis and threefold in K+-depletion. In summary, the hypocitraturia of chronic metabolic acidosis is associated with an increase in ATP citrate lyase activity and protein abundance, and is partly reversed by inhibition of this enzyme. These results suggest an important role for ATP citrate lyase in proximal tubular citrate metabolism.