STRUCTURE AND FUNCTION OF LACTOSE SYNTHASE

α-乳清蛋白(LA)和溶菌酶(LZ)是与 3D结构非常相似，但功能却非常不同。拉，那个 乳糖合成酶的调节蛋白，调节 催化成分，半乳糖基转移酶(GT)，因此它可以催化 乳糖的生物合成，而LZ催化糖苷的水解 细菌细胞壁中的键。La还能与高钙离子结合 亲和力。钙不是活动所必需的，因为活动 在LA分子通过一个 细胞内隔间，不太可能是稳定所必需的。 然而，需要CA2来从减少的 变性的形式。虽然大多数已知的LZ不结合钙，但a 最近发现了与钙结合的亚类，显然是在一个 与洛杉矶的站点对应的站点。在E. Coli作为一种融合蛋白，可以从中制备天然活性LA，我们 建议(I)使用定点突变来检验以下假设 LA的结构-功能关系及其结构基础 与LZ的功能分歧；(Ii)继续结构研究 以及在GT中使用化学程序的行动。独特的交联点将 将其引入LA以便于鉴定结合位点 对于GT上的LA；(Iii)与LA和Ca-进行进一步的复性研究- 结合来自还原变性状态的LZ以确定是否 LA的复性在更广泛的条件下是钙依赖的，并且 阐明这些LZ的折叠特性；(Iv)改变天冬氨酸 LA的钙结合部位的残基决定是否偶联 与钙结合的天然折叠是电荷分布的函数 在钙结合肘部；(V)表征物理性质和 在(I)和(IV)中产生的突变体LAS的稳定性。对于具有 有趣的特性这将涉及对其3D的协作研究 X-射线晶体结构分析；(Vi)鸡LZ基因的表达 并确定类LA序列的功能和结构效应 改变。这是一个长期目标，取决于在 (I)和(Iv)。

Alpha-lactalbumin (LA) and lysozyme (LZ) are homologous proteins with closely similar 3D structures but highly divergent functions. LA, the regulatory protein of lactose synthase, modulates the specificity of the catalytic component, galactosyltransferase (GT) so that it can catalyze the biosynthesis of lactose, whereas LZ catalyzes the hydrolysis of glycosidic bonds in bacterial cell walls. LA also binds a Ca2+ ion with high affinity. The Ca2+ is not required for activity and, because the activity of an LA molecule occurs briefly during its passage through an intracellular compartment, is unlikely to be necessary for stabilization. Ca2+ is, however, required for regenerating native LA from the reduced denatured form. Although the majority of known LZs do not bind Ca2+, a subclass has been recently identified that do bind Ca2+, apparently at a site corresponding to that in LA. Having expressed that cDNA for LA in E. coli as a fusion protein from which native active LA can be prepared, we propose to (i) use site-directed mutagenesis to test hypotheses regarding structure-function relationships in LA, and the structural basis of its functional divergence from LZ; (ii) continue investigations of structure and action in GT using chemical procedures. Unique crosslinking sites will be introduced into LA to facilitate the identification of the binding site for LA on GT; (iii) conduct further refolding studies with LA and Ca2+- binding LZs from the reduced denatured state to determine if the renaturation of LA is Ca2+-dependent over a wider range of conditions and to elucidate the folding properties of these LZs; (iv) change aspartyl residues in the Ca2+-binding site of LA to determine if the coupling of native folding with Ca2+ binding is a function of the charge distribution in the Ca2+-binding elbow; (v) characterize the physical properties and stabilities of mutant LAs generated in (i) and (iv). For mutants with interesting properties this will involve a collaborative study of their 3D structures by X-ray crystallography; (vi) express the cDNA for chicken LZ and to determine the functional and structural effects of LA-like sequence changes. This is a long-term goal that is dependent on results obtained in (i) and (iv).