he authors claim that Ala282 to Val substitution in the active site of MCD chang
ID: 302822 • Letter: H
Question
he authors claim that Ala282 to Val substitution in the active site of MCD changes Omproves substrate catalytic rate. What changes in kinetic parameters allow to reach this conclusion? Table 2 Ala-282 and Phe 284 c to Ala-28s and Phve-257 in PaMCDrspectiwely ND.no Actesctalby 82.3+ 14 RAMCD A282 31.8 ± 0.6 ND ND ND ND ND ND ND RsMCD A2821 RAMCD A28272S4A RMCD A282F/E284L ND ND 071 +a.o3 43949 ND ND ND ND 4D ND A. a decrease in KM and an increase in kcat 0 B. an increase in KM and an increase in kcat ° C. a decrease in KM and a decrease in kcat D. an increase in KM and a decrease in 0 E. None of the aboveExplanation / Answer
Substrate specificity of an enzyme towards different substrates is determined by the affinity the enzyme has for a particular substrate. Substrate affinity is inversely proportional to Km or Michaelis constant. Km is the substrate concentration at which the rate of the reaction is half of Vmax (the maximal rate of the reaction at saturating substrate concentrations). So a smaller value of Km indicates high affinity the enzyme has for the substrate (and vice-versa). This indicates that reactions with a lower Km will reach the Vmax at lower substrate concentrations, compared to reactions with a higher Km value. This can also be interpreted that enzyme with different Km values for two different substrates will have greater specificity/affinity for the substrate which confers a lower Km value. In this case, Ala282Val substitution in the active site of MCD (second row of the table) improves substrate specificity or affinity for succinyl-CoA than (2S)-methylsuccinyl CoA. This is because succinyl-CoA confers a lower Km value (~57), therefore higher affinity than the other substrate (~64). Also when compared to WT enzyme's affinity towards succinyl-CoA, Km has significantly decreased from ~141 to ~57, even less that half of the WT's Km value. However the same is not observed for methyl-succinylCoA (~80 to ~64). [a decrease in Km] Catalytic rate of an enzyme is described by kcat or the turnover number. A higher kcat value indicates more substrate molecules are being turned over per second; while kcat/Km is the catalytic efficiency of the enzyme, and refers to the efficacy of the transformation of the bound substrate to its product. In this case, the catalytic rate or kcat value of the WT and A282V mutant enzyme for succinyl CoA is comparable (~0.32 compared to 0.30), suggesting that the Ala282Val substitution does not affect the catalytic rate of the enzyme for this particular substrate. But when compared to the catalytic rate of A282V enzyme in presence of 2S-methylsuccinyl- CoA, the kcat value in presence of succinyl CoA is significantly reduced (31.8 vs 0.30). This indicates that the mutation affects the catalytic rates of the enzyme in response to different substrates. [decrease in kcat] So the answer will be : C. a decrease in KM and a decrease in kcat
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