Hendrickson T L, Imperiali B
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena 91125, USA.
Biochemistry. 1995 Jul 25;34(29):9444-50. doi: 10.1021/bi00029a020.
Oligosaccharyl transferase activity exhibits an absolute requirement for certain divalent metal cations. Studies with reconstituted enzyme suggest a preference for metal ions that can adopt an octahedral coordination geometry. In order to gain insight into the specific role of the metal cation in catalysis, we have investigated the influence of the metal cofactor on catalytic turnover of the tripeptide substrate Bz-Asn-Leu-Thr-NHMe (1) and a closely related sulfur-containing analog, Bz-Asn(gamma S)-Leu-Thr-NHMe (2). The metal ion substitution studies reveal that 1 is effectively turned over in the presence of several metal ions (Mn2+, Fe2+, Mg2+, and Ca2+). In contrast, 2 is only glycosylated in the presence of the thiophilic metal cations manganese and iron. When the enzyme is reconstituted with the oxophilic cations magnesium and calcium, 2 shows minimal substrate behavior. With the amide substrate 1, the distinct preference for manganese over magnesium may argue against direct coordination of the metal to the lipid-linked substrate pyrophosphate moiety. This fact, together with the comparative studies with asparagine- and thioasparagine-containing tripeptides, implicates the metal cofactor in a role that places it proximal to the peptide binding site.
寡糖基转移酶活性对某些二价金属阳离子表现出绝对需求。对重组酶的研究表明，该酶更倾向于能形成八面体配位几何结构的金属离子。为深入了解金属阳离子在催化过程中的具体作用，我们研究了金属辅因子对三肽底物Bz-Asn-Leu-Thr-NHMe（1）和一种密切相关的含硫类似物Bz-Asn（γS）-Leu-Thr-NHMe（2）催化周转的影响。金属离子替代研究表明，在几种金属离子（Mn2+、Fe2+、Mg2+和Ca2+）存在的情况下，1能有效周转。相比之下，2仅在亲硫金属阳离子锰和铁存在时才发生糖基化。当用亲氧阳离子镁和钙重组该酶时，2表现出最小的底物活性。对于酰胺底物1，锰相对于镁的明显偏好可能表明金属并非直接与脂质连接的底物焦磷酸部分配位。这一事实，连同对含天冬酰胺和硫代天冬酰胺的三肽的比较研究，表明金属辅因子在靠近肽结合位点的位置发挥作用。
