STRUCTURE AND FUNCTION OF LACTOSE SYNTHASE

α-乳白蛋白（LA）和溶菌酶（LZ）是同源蛋白， 非常相似的三维结构，但功能高度不同。 洛杉矶，The 乳糖合成酶的调节蛋白，调节 催化组分，半乳糖基转移酶（GT），使其可以催化 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还结合高浓度的Ca 2+离子， 亲和力 Ca 2+不是活性所必需的，因为活性 LA分子在其通过 细胞内区室，不太可能是稳定所必需的。 然而，Ca 2+是从还原的LA再生天然LA所需的。 变性形式。 虽然大多数已知的LZ不结合Ca 2+， 最近已经鉴定出确实结合Ca 2+的亚类，显然以 与LA中的位置相对应。 在E. 大肠杆菌作为融合蛋白，从天然活性LA可以制备，我们 建议（一）使用定点诱变来测试关于 结构-功能关系及其结构基础 从LZ的功能分歧;（ii）继续调查结构 和GT中使用化学程序的作用。 独特的交联位点将 引入LA以便于识别结合位点 （iii）用LA和Ca 2 +-进行进一步的重折叠研究。 从还原变性状态结合LZ，以确定 LA的复性在较宽的条件范围内是Ca 2+依赖性的， 阐明这些LZ的折叠特性;（iv）改变乙酰基 在LA的Ca 2+结合位点的残基，以确定是否耦合 与Ca 2+结合的天然折叠是电荷分布的函数 在Ca 2+结合肘中;（v）表征物理性质， （i）和（iv）中产生的突变LA的稳定性。 对于突变体， 有趣的属性，这将涉及他们的3D合作研究 （vi）表达鸡LZ的cDNA 并确定LA样序列的功能和结构效应 变化 这是一个长期目标，取决于 (i)及（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).