Mitochondrial malate dehydrogenase (MDH)-citrate synthase (CS) multi-enzyme complex is a part of the Krebs tricarboxylic acid (TCA) cycle ‘metabolon’ which is enzyme machinery catalyzing sequential reactions without diffusion of reaction intermediates into a bulk matrix. This complex is assumed to be a dynamic structure involved in the regulation of the cycle by enhancing metabolic flux. Microscale Thermophoresis analysis of the porcine heart MDH-CS complex revealed that substrates of the MDH and CS reactions, NAD+ and acetyl-CoA, enhance complex association while products of the reactions, NADH and citrate, weaken the affinity of the complex. Oxaloacetate enhanced the interaction only when it was present together with acetyl-CoA. Structural modeling using published CS structures suggested that the binding of these substrates can stabilize the closed format of CS which favors the MDH-CS association. Two other TCA cycle intermediates, ATP, and low pH also enhanced the association of the complex. These results suggest that dynamic formation of the MDH-CS multi-enzyme complex is modulated by metabolic factors responding to respiratory metabolism, and it may function in the feedback regulation of the cycle and adjacent metabolic pathways.
Annonamuricata plant parts possess a broad range of medicinal and biological properties. This research compared the chemical composition and antioxidant properties of Annonamuric at aparts. Proximate, mineral, total phenol and total flavonoid content as well as invitro antioxidant activity were examined. Results revealed the leaves contained significantly(p<0.05) higher composition of moisture (8.69±0.22%), ash (4.60±0.02%), protein (14.53±0.11%), crude fat (10.28±0.03%),chromium (0.38±0.05 mg/100g), nickel (1.75±0.04mg/100g), total phenol (1.01±0.03mgpyrocatechol/mL) and total flavonoid (1.12±0.03mgGAE/mL)compared to the respective values for root. Carbohydrate (9.29±0.24%), lead (0.13±0.02mg/100g) and cobalt (1.93±0.02mg/100g) composition was significantly lower(p<0.05) in the leaves compared to the respective compositions in the root. The leaf and root extract exhibited a concentration-dependent increase in hydroxyl radical scavenging activity with no observable(p<0.05)difference in their EC50 value. This study suggests the leaves of A. muricata found in Covenant University had better chemical composition when compared to the root. Nonetheless, these plant parts may be further exploited for not only their nutritive composition and mineral content but also a natural source of antioxidant agents.
Two tricarboxylic acid (TCA) cycle enzymes, citrate synthase (CS) and malate dehydrogenase (MDH), form a multi‐enzyme complex (MDH‐CS) that catalyzes two sequential reactions of the cycle. It is not known if this complex is static or dynamic and if it is dynamic, what are the factors that regulate its association/dissociation? Various metabolic factors, including the concentrations of substrates and products of the MDH‐CS reaction, TCA cycle intermediates, energy and redox status, and pH, fluctuate with change in metabolic status in the mitochondrial matrix and function as allosteric modulators of the TCA cycle enzymes. Therefore, we hypothesize that these factors regulate the association/dissociation of the MDH‐CS complex. We performed an in vitro study on porcine heart MDH and CS to evaluate the effects of the afore mentioned factors on the affinity of the multi‐enzyme complex by determining equilibrium dissociation constant (Kd) of the multienzyme complex using MicroScale Thermophoresis (MST). We found that substrates of the MDH‐CS reaction, NAD+, acetyl CoA, and malate, enhanced complex association while products of the reaction, NADH and citrate, weakened the binding affinity of the enzymes. Oxaloacetate, which is channeled by this complex, showed no significant effect on the affinity. However, it enhanced the positive effect of acetyl CoA when both compounds were present while oxaloacetate showed no effect on binding affinity when added together with NADH. Succinyl CoA, and α‐ketoglutarate, which proceed from connecting pathways, also enhanced association of the complex. pH did not alter binding affinity. These results support the hypothesis that formation of the MDH‐CS complex is dynamic and modulated by metabolic factors in response to respiratory metabolism.
Mitochondrial malate dehydrogenase (MDH)-citrate synthase (CS) multi-enzyme complex is a part of the Krebs tricarboxylic acid (TCA) cycle ‘metabolon’ which is enzyme machinery catalyzing sequential reactions without diffusion of reaction intermediates into a bulk matrix. This complex is assumed to be a dynamic structure involved in the regulation of the cycle by enhancing metabolic flux. Microscale Thermophoresis analysis of the porcine heart MDH-CS complex revealed that substrates of the MDH and CS reactions, NAD+ and acetyl-CoA, enhance complex association while products of the reactions, NADH and citrate, weaken the affinity of the complex. Oxaloacetate enhanced the interaction only when it was presented together with acetyl-CoA. Structural modeling using published CS structures suggested that the binding of these substrates can stabilize the closed format of CS which favors the MDH-CS association. Two other TCA cycle intermediates, ATP, and low pH also enhanced the association of the complex. These results suggest that dynamic formation of the MDH-CS multi-enzyme complex is modulated by metabolic factors responding to respiratory metabolism, and it may function in the feedback regulation of the cycle and adjacent metabolic pathways.
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