Gao Zhong, Keeling Peter, Shibles Richard, Guan Hanping
Interdepartmental Plant Physiology Program and Agronomy Department, Iowa State, University, Ames, IA, USA.
Arch Biochem Biophys. 2004 Jul 1;427(1):1-7. doi: 10.1016/j.abb.2004.01.010.
It has been suggested that the lysine residue in the conserved K-T-G-G motif could be the substrate ADP-glucose binding site of Escherichia coli glycogen synthase (GS). Since the K-X-G-G motif is highly conserved between E. coli GS and all the maize starch synthase (SS) isozymes, it has become widely accepted that the lysine in the conserved K-T-G-G motif may also function as the ADPGlc binding site of maize SS. We have used chemical modification and site-directed mutagenesis to study the function of lysine residues in SS. Pyridoxal-5'-phosphate inactivated maize SSIIa activity in a time and concentration dependent manner. ADPGlc completely protected SSIIa from inactivation by pyridoxal-5'-phosphate, indicating that lysine residue(s) could be important for ADPGlc binding and enzyme catalysis. In contrast to E. coli GS, mutation of conserved lysine193 (K-T-G-G) in maize SS did not alter the ADPGlc binding while significantly changing the enzyme activity toward different primers. Our results suggest that lysine-193 (K-T-G-G) is not directly involved in ADPGlc binding, instead mutation in the conserved lysine position affected the primer preference.
有人提出，保守的K-T-G-G基序中的赖氨酸残基可能是大肠杆菌糖原合酶（GS）的底物ADP-葡萄糖结合位点。由于K-X-G-G基序在大肠杆菌GS和所有玉米淀粉合酶（SS）同工酶之间高度保守，人们普遍认为保守的K-T-G-G基序中的赖氨酸也可能作为玉米SS的ADPGlc结合位点。我们使用化学修饰和定点诱变来研究SS中赖氨酸残基的功能。磷酸吡哆醛以时间和浓度依赖的方式使玉米SSIIa失活。ADPGlc完全保护SSIIa不被磷酸吡哆醛失活，表明赖氨酸残基可能对ADPGlc结合和酶催化很重要。与大肠杆菌GS不同，玉米SS中保守的赖氨酸193（K-T-G-G）突变不会改变ADPGlc结合，同时显著改变酶对不同引物的活性。我们的结果表明，赖氨酸-193（K-T-G-G）不直接参与ADPGlc结合，相反，保守赖氨酸位置的突变影响了引物偏好。
