In vitro mutagenesis studies at the arginine residues of adenylate kinase. A revised binding site for AMP in the x-ray-deduced model

Abstract

Although X-ray crystallographic and NMR studies have been made on the adenylate kinases, the substrate-binding sites are not unequivocally established. In an attempt to shed light on the binding sites for MgATP2- and for AMP2- in human cytosolic adenylate kinase (EC 2.7.4.3, hAK1), we have investigated the enzymic effects of replacement of the arginine residues (R44, R132, R138, and R149), which had been assumed by Pai et al. [Pai, E. F., Sachsenheimer, W., Schirmer, R. H., & Schulz, G. E. (1977) J. Mol. Biol. 114, 37-45] to interact with the phosphoryl groups of AMP2- and MgATP2-. With use of the site-directed mutagenesis method, point mutations were made in the artificial gene for hAK1 [Kim, H. J., Nishikawa, S., Tanaka, T., Uesugi, S., Takenaka, H., Hamada, M., & Kuby, S. A. (1989) Protein Eng. 2, 379-386] to replace these arginine residues with alanyl residues and yield the mutants R44A hAK1, R132A hAK1, R138A hAK1, and R149A hAK1. The resulting large increases in the Km,app values for AMP2- of the mutant enzymes, the relatively small increases in the Km,app values for MgATP2-, and the fact that the R132A, R138A, and R149A mutant enzymes proved to be very poor catalysts are consistent with the idea that the assigned substrate binding sites of Pai et al. (1977) have been reversed and that their ATP-binding site may be assigned as the AMP site.

Adenylate kinase fromStreptococcus pneumoniaeis essential for growth through its catalytic activity

“…Among the mutated proteins tested, mutation at Arg‐89 most significantly deteriorated the activity. We tested both R89A and R89G, because previous studies suggested that mutating Arg to Ala or Gly was efficient, in reducing the catalytic activity [17,18]. For SpAdK, both R89A and R89G exhibited comparable reduction in activity.…”

Structural Characterization of the Closed Conformation of Mouse Guanylate Kinase

“…In the mGMPK GMP-ADP structure, Arg 137 interacts with the ␣-and ␤-phosphates of ADP, and Arg 148 interacts with the GMP phosphate. The analogous arginines in adenylate kinase have been shown by mutational analysis to be essential for catalysis (30). Additionally, structural studies performed with Dictyostelium discoideum uridylate kinase have also demonstrated the importance of these LID arginines (29,31).…”

Structure and Mechanism of F 0 F 1 ‐Type ATP Synthases and ATPases

“…Porcine adenylate kinase arginyls at equivalent positions in the sequence to bovine F1 Arg-295 and Lys-300 have been mutated (R132A, R138A) with a resultant large increase in the K, for AMP (100). In addition, an arginyl of yeast adenylate kinase (Arg-165) in an equivalent position in the sequence to bovine Lys-300 is seen in the crystal structure to make contact with phosphoryl groups (101).…”