Glyoxylate Carboligase: A Unique Thiamin Diphosphate-Dependent Enzyme That Can Cycle between the 4鈥?Aminopyrimidinium and 1鈥?4鈥?Iminopyrimidine Tautomeric Forms in the Absence of the Conserved Glutama

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• 作者：Natalia Nemeria ; Elad Binshtein ; Hetalben Patel ; Anand Balakrishnan ; Ilan Vered ; Boaz Shaanan ; Ze鈥檈v Barak ; David Chipman ; Frank Jordan
• 刊名：Biochemistry
• 出版年：2012
• 出版时间：October 9, 2012
• 年：2012
• 卷：51
• 期：40
• 页码：7940-7952
• 全文大小：695K
• 年卷期：v.51,no.40(October 9, 2012)
• ISSN：1520-4995

文摘

Glyoxylate carboligase (GCL) is a thiamin diphosphate (ThDP)-dependent enzyme, which catalyzes the decarboxylation of glyoxylate and ligation to a second molecule of glyoxylate to form tartronate semialdehyde (TSA). This enzyme is unique among ThDP enzymes in that it lacks a conserved glutamate near the N1鈥?atom of ThDP (replaced by Val51) or any other potential acid鈥揵ase side chains near ThDP. The V51D substitution shifts the pH optimum to 6.0鈥?.2 (pK_a of 6.2) for TSA formation from pH 7.0鈥?.7 in wild-type GCL. This pK_a is similar to the pK_a of 6.1 for the 1鈥?4鈥?iminopyrimidine (IP)鈥?鈥?aminopyrimidinium (APH⁺) protonic equilibrium, suggesting that the same groups control both ThDP protonation and TSA formation. The key covalent ThDP-bound intermediates were identified on V51D GCL by a combination of steady-state and stopped-flow circular dichroism methods, yielding rate constants for their formation and decomposition. It was demonstrated that active center variants with substitution at I393 could synthesize (S)-acetolactate from pyruvate solely, and acetylglycolate derived from pyruvate as the acetyl donor and glyoxylate as the acceptor, implying that this substitutent favored pyruvate as the donor in carboligase reactions. Consistent with these observations, the I393A GLC variants could stabilize the predecarboxylation intermediate analogues derived from acetylphosphinate, propionylphosphinate, and methyl acetylphosphonate in their IP tautomeric forms notwithstanding the absence of the conserved glutamate. The role of the residue at the position occupied typically by the conserved Glu controls the pH dependence of kinetic parameters, while the entire reaction sequence could be catalyzed by ThDP itself, once the APH⁺ form is accessible.