Plater A R, Zgiby S M, Thomson G J, Qamar S, Wharton C W, Berry A
School of Biochemistry and Molecular Biology, University of Leeds, Leeds, LS2 9JT, England.
J Mol Biol. 1999 Jan 15;285(2):843-55. doi: 10.1006/jmbi.1998.2376.
The two classes of fructose-1,6-bisphosphate aldolase both catalyse the reversible cleavage of fructose 1,6-bisphosphate into dihydroxyacetone phosphate and glyceraldehyde 3-phosphate. The Class I aldolases use Schiff base formation as part of their catalytic mechanism, whereas the Class II enzymes are zinc-containing metalloproteins. The mechanism of the Class II enzymes is less well understood than their Class I counterparts. We have combined sequence alignments of the Class II family of enzymes with examination of the crystal structure of the enzyme to highlight potentially important aspartate and asparagine residues in the enzyme mechanism. Asp109, Asp144, Asp288, Asp290, Asp329 and Asn286 were targeted for site-directed mutagenesis and the resulting proteins purified and characterised by steady-state kinetics using either a coupled assay system to study the overall cleavage reaction or using the hexacyanoferrate (III) oxidation of the enzyme bound intermediate carbanion to investigate partial reactions. The results showed only minor changes in the kinetic parameters for the Asp144, Asp288, Asp290 and Asp329 mutants, suggesting that these residues play only minor or indirect roles in catalysis. By contrast, mutation of Asp109 or Asn286 caused 3000-fold and 8000-fold decreases in the kcat of the reaction, respectively. Coupled with the kinetics measured for the partial reactions the results clearly demonstrate a role for Asn286 in catalysis and in binding the ketonic end of the substrate. Fourier transform infra-red spectroscopy of the wild-type and mutant enzymes has further delineated the role of Asp109 as being critically involved in the polarisation of the carbonyl group of glyceraldehyde 3-phosphate.
两类1,6-二磷酸果糖醛缩酶均催化1,6-二磷酸果糖可逆裂解为磷酸二羟丙酮和3-磷酸甘油醛。I类醛缩酶在其催化机制中利用席夫碱的形成，而II类酶是含锌金属蛋白。II类酶的机制相比其I类对应物了解得较少。我们将II类酶家族的序列比对与该酶晶体结构的研究相结合，以突出酶机制中潜在重要的天冬氨酸和天冬酰胺残基。天冬氨酸109、天冬氨酸144、天冬氨酸288、天冬氨酸290、天冬氨酸329和天冬酰胺286被作为定点诱变的目标，所得蛋白质经纯化，并通过稳态动力学进行表征，使用偶联测定系统研究整体裂解反应，或使用酶结合的中间体碳负离子的高铁氰酸盐（III）氧化来研究部分反应。结果表明，天冬氨酸144、天冬氨酸288、天冬氨酸290和天冬氨酸329突变体的动力学参数仅有微小变化，表明这些残基在催化中仅起次要或间接作用。相比之下，天冬氨酸109或天冬酰胺286的突变分别导致反应的催化常数降低3000倍和8000倍。结合部分反应测得的动力学，结果清楚地证明天冬酰胺286在催化和结合底物的酮基末端中起作用。野生型和突变型酶的傅里叶变换红外光谱进一步阐明了天冬氨酸109的作用，即关键参与3-磷酸甘油醛羰基的极化。
