Bell J E, Beyer T A, Hill R L
J Biol Chem. 1976 May 25;251(10):3003-13.
Initial rate parameters obtained with bovine galactosyltransferase at saturating Mn2+ concentrations, and a variety of acceptors including N-acetylglucosamine, glucose, ovalbumin, and di-N-acetylglucosamine are inconsistent with an ordered addition of UDP-galactose and acceptor substrates to the enzyme-Mn2+ complex. Inhibition patterns with N-acetylglucosamine or UDP-glucose as inhibitors of the galactosylation of ovalbumin indicated that either UDP-galactose or N-acetylglucosamine can bind to an enzyme-Mn2+ complex by a random equilibrium mechanism. Initial rate studies also indicate that alpha-lactalbumin may bind to either an enzyme-Mn2+-acceptor complex or an enzyme-Mn2+-UDP-galactose complex, suggesting that lactose synthesis also proceeds by a random equilibrium addition of substrates and alpha-lactalbumin. From the initial rate data assuming the random equilibrium mechanism, the dissociation constants for UDP-galactose, acceptor substrates, and alpha-lactalbumin from the appropriate complexes have been calculated. These values are in good agreement with those obtained independently by nonkinetic methods, providing additional support for the proposed random equilibrium mechanism. From similar studies with a cross-linked complex of alpha-lactalbumin and transferase, dissociation constants for UDP-galactose and acceptor substrates from the enzyme-Mn2+-alpha-lactalbumin complex were calculated. Comparison of each of the dissociation constants in the substrate addition phase shows that the binding of acceptor substrates and alpha-lactalbumin to enzyme-Mn2+ complexes is highly synergistic; the affinity of alpha-lactalbumin for the enzyme-Mn2+ acceptor complex is about 2 orders of magnitude greater than for the enzyme-Mn2+ complex. Similarly, the affinity of the acceptor for the enzyme-Mn2+-alpha-lactalbumin complex is about 2 orders of magnitude greater than the enzyme-Mn2+ complex. Synergism is also observed between alpha-lactalbumin and UDP-galactose binding but the synergism is much less than that observed with acceptor substrates and alpha-lactalbumin. Thus, the large decrease in the Michaelis constant for glucose in the presence of alpha-lactalbumin, which is observed for lactose synthesis by the galactosyltranferase, is primarily the result of the high degree of synergism in the binding of alpha-lactalbumin and glucose to enzyme-Mn2+ complexes. This synergism also accounts for the activation of N-acetyllactosamine synthesis by alpha-lactalbumin at low concentrations (less than 2 mM) of N-acetylglucosamine. An abortive enzyme-Mn2+-UDP-acceptor complex in the product release phase of the reaction appears to account for the inhibition of either lactose, or N-acetyllactosamine synthesis at a high concentration of either N-acetylglucosamine or glucose. This abortive complex is further stabilized by alpha-lactalbumin, thus the resulting substrate inhibition is observed at much lower acceptor concentrations in the presence of alpha-lactalbumin.
在饱和Mn2+浓度下，使用牛半乳糖基转移酶以及包括N - 乙酰葡糖胺、葡萄糖、卵清蛋白和二 - N - 乙酰葡糖胺在内的多种受体所获得的初始速率参数，与UDP - 半乳糖和受体底物按顺序添加到酶 - Mn2+复合物的情况不一致。以N - 乙酰葡糖胺或UDP - 葡萄糖作为卵清蛋白半乳糖基化的抑制剂时的抑制模式表明，UDP - 半乳糖或N - 乙酰葡糖胺可以通过随机平衡机制与酶 - Mn2+复合物结合。初始速率研究还表明，α - 乳白蛋白可能与酶 - Mn2+ - 受体复合物或酶 - Mn2+ - UDP - 半乳糖复合物结合，这表明乳糖合成也是通过底物和α - 乳白蛋白的随机平衡添加进行的。根据假设的随机平衡机制的初始速率数据，已计算出UDP - 半乳糖、受体底物和α - 乳白蛋白从相应复合物中的解离常数。这些值与通过非动力学方法独立获得的值非常一致，为所提出的随机平衡机制提供了额外支持。通过对α - 乳白蛋白和转移酶的交联复合物进行类似研究，计算出了酶 - Mn2+ - α - 乳白蛋白复合物中UDP - 半乳糖和受体底物的解离常数。底物添加阶段中每个解离常数的比较表明，受体底物和α - 乳白蛋白与酶 - Mn2+复合物的结合具有高度协同性；α - 乳白蛋白对酶 - Mn2+受体复合物的亲和力比对酶 - Mn2+复合物的亲和力大约高2个数量级。同样，受体对酶 - Mn2+ - α - 乳白蛋白复合物的亲和力比对酶 - Mn2+复合物的亲和力大约高2个数量级。在α - 乳白蛋白和UDP - 半乳糖结合之间也观察到协同作用，但这种协同作用远小于受体底物和α - 乳白蛋白之间的协同作用。因此，在半乳糖基转移酶催化乳糖合成过程中，在α - 乳白蛋白存在下观察到的葡萄糖米氏常数的大幅降低，主要是α - 乳白蛋白和葡萄糖与酶 - Mn2+复合物结合时高度协同作用的结果。这种协同作用也解释了在低浓度（小于2 mM）N - 乙酰葡糖胺时α - 乳白蛋白对N - 乙酰乳糖胺合成的激活作用。反应产物释放阶段中的无效酶 - Mn2+ - UDP - 受体复合物似乎解释了在高浓度N - 乙酰葡糖胺或葡萄糖存在下对乳糖或N - 乙酰乳糖胺合成的抑制作用。这种无效复合物被α - 乳白蛋白进一步稳定，因此在α - 乳白蛋白存在下，在低得多的受体浓度下就观察到了由此产生的底物抑制作用。
