Enzyme synthesis in the regulation of hepatic `malic'' enzyme activity

A homogeneous preparation of ;malic' enzyme (EC 1.1.1.40) from livers of thyroxine-treated rats was used to prepare in rabbits an antiserum to the enzyme that reacts monospecifically with the ;malic' enzyme in livers of rats in several physiological states. Changes in enzyme activity resulting from modification of the state of the animal are hence due to an altered amount of enzyme protein. The antiserum has been used to precipitate out ;malic' enzyme from heat-treated supernatant preparations of livers from both adult and neonatal rats, in a number of physiological conditions, that had been injected 30min earlier with l-[4,5-(3)H]leucine. The low incorporations of radioactivity into the immunoprecipitable enzyme have permitted the qualitative conclusion that changed enzyme activity in adult rats arises mainly from alterations in the rate of enzyme synthesis. The marked increase in ;malic' enzyme activity that occurs naturally or as a result of thyroxine treatment of the weanling rat is likewise due to a marked increase in the rate of enzyme synthesis possibly associated with a concurrent diminished rate of enzyme degradation.     

Regulation of `malic'' enzyme of Solanum tuberosum by metabolites

A purification of ;malic' enzyme from potato is described. The purified enzyme is specific for NADP and requires a bivalent cation for activity. At pH values below 7 the plot of rate versus malate concentration approximates to normal Michaelis-Menten kinetics. At pH values above 7 the plot of rate versus malate concentration is sigmoid. A number of dicarboxylic acids activate the enzyme and remove the sigmoidicity. The enzyme is inhibited by phosphate, triose phosphates and AMP. In general, effectors of the oxidative decarboxylation of malate behave in the same manner in the reductive carboxylation of pyruvate. The response of the enzyme to energy charge is reported and the physiological significance of the response to metabolites is discussed in relation to the proposed role of the enzyme in the control of pH.     

The energetics of intramolecular reactions and enzyme catalysis.

The relative rates of reactions should always be examined by an awareness of differential effects. The magnitude and variation of the relative rates of intramolecular reactions can be rationalized by the differences in entropy and strain energy. The relative rates of enzyme-catalysed reactions are sometimes due to groundstate effects. The beta-lactamase-catalysed hydrolysis of beta-lactam antibiotics may require a unique disposition of catalytic groups owing to an unusual process of bond fission in the four membered ring.     

Citrate-cleavage enzyme, `malic'' enzyme and certain dehydrogenases in embryonic and growing chicks

The activities of several enzymes possibly implicated in lipogenesis were measured in the soluble fraction of homogenates of liver and adipose tissue of embryonic and growing chicks. The activities of adipose-tissue enzymes showed little or no change. The activities of hepatic hexose monophosphate-shunt dehydrogenases, malate dehydrogenase, 3-phosphoglyceraldehyde dehydrogenase and NAD-linked α-glycerophosphate dehydrogenase also showed little or no change. Isocitrate dehydrogenase activity in liver rose to a peak on the day of hatching and fell to half the peak value during the next 12 days, where it remained to 26 days after hatching. The activities of `malic' enzyme and citrate-cleavage enzyme showed very low stable values in embryonic liver and remarkable rises during the early part of the post-hatching period. An 85-fold increase in the activity of `malic' enzyme activity was completed in 7 days and a 15-fold increase in that of citrate-cleavage enzyme in 5 days. The activities then attained were maintained up to 26 days after hatching. 2. The increases in the activities of hepatic citrate-cleavage enzyme and `malic' enzyme occurred simultaneously with a marked increase in lipogenesis, suggesting a relationship of these enzymes to lipogenesis in chick liver. By contrast, activity of the hexose monophosphate-shunt dehydrogenases does not appear to be thus associated.     

Isolation and properties of a `malic'' enzyme from cauliflower bud mitochondria

1. A ;malic' enzyme [l-malate-NAD oxidoreductase (decarboxylating), EC 1.1.1.39] has been isolated from cauliflower bud mitochondria and partially purified. 2. The enzyme is specific for l-malate and has an absolute requirement for either Mn(2+), Co(2+) or Mg(2+). 3. The enzyme shows activity with both NAD(+) and NADP(+), but NAD(+) is the preferred cofactor. 4. No appreciable oxaloacetate decarboxylase activity is present in the enzyme preparations even at low pH values. 5. The enzyme is inhibited by NADH and by oxaloacetate and stimulated by SO(4) (2-) and by low concentrations of CoA. 6. The regulatory properties of the enzyme support the proposed role of the enzyme in the utilization of tricarboxylic acid-cycle acids for energy production when glycolysis is suppressed.     

Reduction of α-oxo carboxyylic acids by pigeon liver `malic'' enzyme

1. Pigeon liver ;malic' enzyme [l-malate-NADP(+) oxidoreductase (decarboxylating); EC 1.1.1.40] was shown to catalyse the reductase reaction: [Formula: see text] l-Malate was identified as the reaction product, and was formed in stoicheiometric amount. 2. In addition to oxaloacetate and pyruvate, a number of other alpha-oxo carboxylic acids were also reduced.     

Allostery in enzyme catalysis

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The mechanisms of reductive carboxylation reactions. Carbon dioxide or bicarbonate as substrate of nicotinamide-adenine dinucleotide phosphate-linked isocitrate dehydrogenase and `malic'' enzyme

1. A simple kinetic method was devised to show whether dissolved CO(2) or HCO(3)- ion is the substrate in enzyme-catalysed carboxylation reactions. 2. The time-course of the reductive carboxylation of 2-oxoglutarate by NADPH, catalysed by isocitrate dehydrogenase, was studied by a sensitive fluorimetric method at pH7.3 and pH6.4, with large concentrations of substrate and coenzyme and small carbon dioxide concentrations. 3. Reaction was initiated by the addition of carbon dioxide in one of three forms: (i) as the dissolved gas in equilibrium with bicarbonate; (ii) as unbuffered bicarbonate solution; (iii) as the gas or as an unbuffered solution of the gas in water. Different progress curves were obtained in the three cases. 4. The results show that dissolved CO(2) is the primary substrate of the enzyme, and that HCO(3)- ion is at best a very poor substrate. The progress curves are in quantitative agreement with this conclusion and with the known rates of the reversible hydration of CO(2) under the conditions of the experiments. The effects of carbonic anhydrase confirm the conclusions. 5. Similar experiments on the reductive carboxylation of pyruvate catalysed by the ;malic' enzyme show that dissolved CO(2) is the primary substrate of this enzyme also. 6. The results are discussed in relation to the mechanisms of these enzymes, and the effects of pH on the reactions. 7. The advantages of the method and its possible applications to other enzymes involved in carbon dioxide metabolism are discussed.     

Age-related effects in enzyme catalysis

Summary Rat muscle glyceraldehyde-3-phosphate dehydrogenase is one of several enzymes which have been found to undergo age-related modifications. While the amount of this enzyme in muscle tissue does not change with age, both its specific activity and affinity towards its co-enzyme are significantly reduced in the old tissue.Age-related structural changes were found to exist in the nicotinamide binding site of the enzyme and the reactions leading to the activity loss in old glyceraldehyde-3-phosphate dehydrogenase were shown to involve a reversible modification of the essential cysteine-149 residue at the active site of the enzyme. The aging effects were simulated by a controlled oxidation of cys-149 in samples of young glyceraldehyde-3-phosphate dehydrogenase and subsequent reduction of this residue by 2-mercaptoethanol. The enzyme modified in this way closely resembles native old glyceraldehyde-3-phosphate dehydrogenase, indicating that the structural modifications in the latter enzyme are indeed introduced by a post-translational process. The mechanism for aging of glyceraldehyde-3-phosphate dehydrogenase which is proposed, based on these observations, thus assumes an oxidation of cys-149 as its first step followed by irreversible conformational changes in the enzyme molecule. The aging of glyceraldehyde-3-phosphate dehydrogenase may thus be triggered by the reduced ability of old muscle tissue to protect its constituents against oxidation.Abbreviations CPL circular polarization of luminescence - DTNB 5,5-dithiobis (2-nitrobenzoic acid) - GPDH D-glyceraldehyde-3-phosphate dehydrogenase - ENAD⁺ nicotinamide 1,N⁶-ethenoadenine dinucleotide     

Free-energy landscape of enzyme catalysis

The concept is developed that enzyme mechanisms should be viewed as "catalytic networks" with multiple conformations that occur serially and in parallel in the mechanism. These coupled ensembles of conformations require a multi-dimensional standard free-energy surface that is very "rugged", containing multiple minima and transition states. Experimental and theoretical evidence is presented to support this concept.     

Multiple conformational changes in enzyme catalysis

Understanding the molecular mechanisms of enzyme catalysis and allosteric regulation has been a primary goal of biochemistry for many years. The dynamics of these processes, approached through a variety of kinetic methods, are discussed. The results obtained for many different enzymes suggest that multiple intermediates and conformations are general characteristics of the catalytic process and allosteric regulation. Ribonuclease, dihydrofolate reductase, chymotrypsin, aspartate aminotransferase, and aspartate transcarbamoylase are considered as specific examples. Typical and maximum rates of conformational changes and catalysis are also discussed, based on results obtained from model systems. The nature and rates of interconversion of the intermediates, along with structural information, can be used as the bases for understanding the incredible catalytic efficiency of enzymes. Potential roles of conformational changes in the catalytic process are discussed in terms of static and environmental effects, and in terms of dynamic coupling within the enzyme-substrate complex.