Thermodynamic Analysis Shows Conformational Coupling and Dynamics Confer Substrate Specificity in Fructose-1,6-bisphosphate Aldolase

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• 作者：John A. Pezza ; Jack D. Stopa ; Elizabeth M. Brunyak ; Karen N. Allen ; Dean R. Tolan
• 刊名：Biochemistry
• 出版年：2007
• 出版时间：November 13, 2007
• 年：2007
• 卷：46
• 期：45
• 页码：13010 - 13018
• 全文大小：258K
• 年卷期：v.46,no.45(November 13, 2007)
• ISSN：1520-4995

文摘

Conformational flexibility is emerging as a central theme in enzyme catalysis. Thus, identifyingand characterizing enzyme dynamics are critical for understanding catalytic mechanisms. Herein, couplinganalysis, which uses thermodynamic analysis to assess cooperativity and coupling between distal regionson an enzyme, is used to interrogate substrate specificity among fructose-1,6-(bis)phosphate aldolase(aldolase) isozymes. Aldolase exists as three isozymes, A, B, and C, distinguished by their unique substratepreferences despite the fact that the structures of the active sites of the three isozymes are nearly identical.While conformational flexibility has been observed in aldolase A, its function in the catalytic reaction ofaldolase has not been demonstrated. To explore the role of conformational dynamics in substrate specificity,those residues associated with isozyme specificity (ISRs) were swapped and the resulting chimeras weresubjected to steady-state kinetics. Thermodynamic analyses suggest cooperativity between a terminal surfacepatch (TSP) and a distal surface patch (DSP) of ISRs that are separated by >8.9 Å. Notably, the couplingenergy (G_I) is anticorrelated with respect to the two substrates, fructose 1,6-bisphosphate and fructose1-phosphate. The difference in coupling energy with respect to these two substrates accounts for ~70%of the energy difference for the ratio of k_cat/K_m for the two substrates between aldolase A and aldolase B.These nonadditive mutational effects between the TSP and DSP provide functional evidence that couplinginteractions arising from conformational flexibility during catalysis are a major determinant of substratespecificity.