Liu F, Dong Q, Fromm H J
Department of Biochemistry and Biophysics, Iowa State University, Ames 50011.
J Biol Chem. 1992 Feb 5;267(4):2388-92.
Adenylosuccinate synthetases from different sources contain an N-terminal glycine-rich sequence GDEGKGK, which is homologous to the conserved sequence GXXXXGK found in many other guanine nucleotide-binding proteins or enzymes. To determine the role of this sequence in the structure and function of Escherichia coli adenylosuccinate synthetase, site-directed mutagenesis was performed to generate five mutant enzymes: G12V (Gly12----Val), G15V (Gly15----Val), G17V (Gly17----Val), K18R (Lys18----Arg), and I19T (Ile19----Thr). Comparison of the kinetic properties of the wild-type enzyme and those of the mutant enzymes revealed that the sequence is critical for enzyme activity. Replacement of Gly12, Gly15, or Gly17 with Val, or replacement of Lys18 with Arg, resulted in significant decreases in the kcat/Km values of the enzyme. Because the consensus sequence GXXXXGK(T/S) has been found in many GTP-binding proteins, isoleucine at position 19 in the E. coli adenylosuccinate synthetase was changed to threonine to produce the sequence GDEGKGKT. This mutation, which more closely resembles the consensus sequence, resulted in a 160-fold increase in the Km value for substrate GTP; however, there were no great changes for the other two substrates, IMP and aspartate. Based on these data, we suggest that the N-terminal glycinerich sequence in E. coli adenylosuccinate synthetase plays a more important role in enzyme catalysis than in substrate binding. In addition, a hydrophobic amino acid residue such as isoleucine, leucine, or valine, rather than threonine, may play a critical role in GTP binding in adenosuccinate synthetase. These findings suggest that the glycine-rich sequence in adenylosuccinate synthetase functions differently relative to those in other GTP binding proteins or enzymes.
来自不同来源的腺苷酸琥珀酸合成酶含有一个富含甘氨酸的N端序列GDEGKGK，它与许多其他鸟嘌呤核苷酸结合蛋白或酶中发现的保守序列GXXXXGK同源。为了确定该序列在大肠杆菌腺苷酸琥珀酸合成酶的结构和功能中的作用，进行了定点诱变以产生五种突变酶：G12V（甘氨酸12突变为缬氨酸）、G15V（甘氨酸15突变为缬氨酸）、G17V（甘氨酸17突变为缬氨酸）、K18R（赖氨酸18突变为精氨酸）和I19T（异亮氨酸19突变为苏氨酸）。野生型酶和突变酶的动力学性质比较表明，该序列对酶活性至关重要。用缬氨酸取代甘氨酸12、甘氨酸15或甘氨酸17，或用精氨酸取代赖氨酸18，导致酶的kcat/Km值显著降低。由于在许多GTP结合蛋白中都发现了一致序列GXXXXGK(T/S)，因此将大肠杆菌腺苷酸琥珀酸合成酶中第19位的异亮氨酸改为苏氨酸，以产生序列GDEGKGKT。这种更接近一致序列的突变导致底物GTP的Km值增加了160倍；然而，其他两种底物IMP和天冬氨酸没有太大变化。基于这些数据我们认为，大肠杆菌腺苷酸琥珀酸合成酶中的N端富含甘氨酸序列在酶催化中比在底物结合中起更重要的作用。此外，疏水性氨基酸残基如异亮氨酸、亮氨酸或缬氨酸，而不是苏氨酸，可能在腺苷酸琥珀酸合成酶的GTP结合中起关键作用。这些发现表明，腺苷酸琥珀酸合成酶中富含甘氨酸的序列与其他GTP结合蛋白或酶中的序列功能不同。
