Frey P A
Institute for Enzyme Research, University of Wisconsin-Madison 53705,USA.
FASEB J. 1996 Mar;10(4):461-70.
The biological interconversion of galactose and glucose takes place only by way of the Leloir pathway and requires the three enzymes galactokinase, galactose-1-P uridylyltransferase, and UDP-galactose 4-epimerase. The only biological importance of these enzymes appears to be to provide for the interconversion of galactosyl and glucosyl groups. Galactose mutarotase also participates by producing the galactokinase substrate alpha-D-galactose from its beta-anomer. The galacto/gluco configurational change takes place at the level of the nucleotide sugar by an oxidation/reduction mechanism in the active site of the epimerase NAD+ complex. The nucleotide portion of UDP-galactose and UDP-glucose participates in the epimerization process in two ways: 1) by serving as a binding anchor that allows epimerization to take place at glycosyl-C-4 through weak binding of the sugar, and 2) by inducing a conformational change in the epimerase that destabilizes NAD+ and increases its reactivity toward substrates. Reversible hydride transfer is thereby facilitated between NAD+ and carbon-4 of the weakly bound sugars. The structure of the enzyme reveals many details of the binding of NAD+ and inhibitors at the active site. The essential roles of the kinase and transferase are to attach the UDP group to galactose, allowing for its participation in catalysis by the epimerase. The transferase is a Zn/Fe metalloprotein, in which the metal ions stabilize the structure rather than participating in catalysis. The structure is interesting in that it consists of single beta-sheet with 13 antiparallel strands and 1 parallel strand connected by 6 helices. The mechanism of UMP attachment at the active site of the transferase is a double displacement, with the participation of a covalent UMP-His 166-enzyme intermediate in the Escherichia coli enzyme. The evolution of this mechanism appears to have been guided by the principle of economy in the evolution of binding sites.
半乳糖和葡萄糖的生物相互转化仅通过勒洛伊尔途径进行，并且需要三种酶，即半乳糖激酶、1-磷酸半乳糖尿苷酰转移酶和UDP-半乳糖4-差向异构酶。这些酶唯一的生物学重要性似乎是为半乳糖基和葡萄糖基的相互转化提供条件。半乳糖变旋酶也参与其中，它从β-异头物产生半乳糖激酶的底物α-D-半乳糖。半乳糖/葡萄糖构型的变化通过差向异构酶NAD⁺复合物活性位点中的氧化/还原机制在核苷酸糖水平上发生。UDP-半乳糖和UDP-葡萄糖的核苷酸部分以两种方式参与差向异构化过程：1）作为结合锚，通过糖的弱结合使差向异构化在糖基-C-4处发生；2）通过诱导差向异构酶的构象变化，使NAD⁺不稳定并增加其对底物的反应性。从而促进了NAD⁺与弱结合糖的碳-4之间可逆的氢化物转移。该酶的结构揭示了活性位点处NAD⁺和抑制剂结合的许多细节。激酶和转移酶的基本作用是将UDP基团连接到半乳糖上，使其能够参与差向异构酶的催化作用。转移酶是一种锌/铁金属蛋白，其中金属离子稳定结构而非参与催化。该结构很有趣，它由一个单β-折叠组成，有13条反平行链和1条平行链，由6个螺旋连接。在大肠杆菌的酶中，UMP在转移酶活性位点的附着机制是双取代，涉及共价UMP-组氨酸166-酶中间体的参与。这种机制的进化似乎受到结合位点进化中的经济性原则的指导。
