ATP-binding site of adenylate kinase: mechanistic implications of its homology with ras-encoded p21, F1-ATPase, and other nucleotide-binding proteins.

Abstract

The MgATP binding site of adenylate kinase, located by a combination of NMR and x-ray diffraction, is near three protein segments, five to seven amino acids in length, that are homologous in sequence to segments found in other nucleotide-binding phosphotransferases, such as myosin and Fl-ATPase, ras p21 and transducin GTPases, and cAMPdependent and src protein kinases, suggesting equivalent mechanistic roles of these segments in all of these proteins. Segment 1 is a glycine-rich flexible loop that, on adenylate kinase, may control access to the ATP-binding site by changing its conformation. Segment 2 is an a-helix containing two hydrophobic residues that interact with the adenine-ribose moiety of ATP, and a lysine that may bind to the j8-and y-phosphates of ATP. Segment 3 is a hydrophobic strand of parallel a6-pleated sheet, terminated by a carboxylate, that flanks the triphosphate binding site. The various reported mutations of ras p21 that convert it to a transforming agent all appear to involve segment 1, and such substitutions may alter the properties of p21 by hindering a conformational change at this segment. In F1-ATPase, the flexible loop may, by its position, control both the accessibility and the ATP/ADP equilibrium constant on the enzyme.Adenylate kinase catalyzes the reversible transfer of a phosphoryl group from MgATP to AMP.MgATP + AMP MgADP + ADP.[1]The enzyme has been purified from many sources, the x-ray structure of the porcine muscle enzyme has been reported at 3 A resolution (1), and amino acid sequences have been determined for muscle adenylate kinase from pig (2), human (3), calf, and rabbit (4). It was noted by Walker et al. (5) that two portions of the adenylate kinase sequence had homologous counterparts in the sequences of several ATPases. One of these homologous segments was later found to be present in p21, the GTPase that is the product of the ras oncogene (6-8). The recent finding that a mutation in this segment is the basis of the transforming ability of this protein (9, 10) has amplified the importance of determining the functional role of this segment. We have identified a third homologous region in several of these proteins and have further expanded the list of proteins with sequence homologies to adenylate kinase. Through a series of NMR studies on porcine (11) and rabbit muscle (12) High-field proton NMR was used to study the interaction of metal-ATP substrates with porcine (11) and rabbit muscle adenylate kinase (12), and with a globular peptide fragment of the latter enzyme consisting of residues 1-45 that binds metal-ATP with comparable affinity (13). Paramagnetic effects of B,y-bidentate Cr3+-ATP on the relaxation rates of protons of the enzyme and the peptide were measured and provided a total of eight distances from Cr3+ to the side chains of specific amino acid residues. Time-dependent nuclear tIn some of the proteins of (32). 907The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "adv...

1H‐NMR studies on nucleotide binding to the catalytic sites of bovine mitochondrial F1‐ATPase

“…1). The large size of the TRNOE between Table 1 Normalized It is of interest to recall that adenine nucleotides have also been shown to bind in an anti conformation on several enzymes including sarcoplasmic reticulum Ca 2÷ ATPase, muscle adenylate kinase, pyruvate kinase and creatine kinase [13][14][15][16].…”

Agrobacterium tumefaciens VirB11 protein requires a consensus nucleotide-binding site for function in virulence

“…Although nucleotide binding to the fusion protein could not be detected, the predicted amino acid sequence of VirB11 contains a conserved sequence (GPTGSGKT) which corresponds to the Walker box A nucleotide-binding site (GxxGxGKT/S) in many ATP-binding proteins (63). The amino acids of this conserved region have been shown to be important for binding nucleotides in these proteins by nuclear magnetic resonance (17)(18)(19), X-ray crystallography (24,55,69), and genetic (23,49,60) studies. The highly conserved lysine is thought to function by interacting with the ␥-phosphate of ATP (17-19, 55, 69), and changes at this position greatly reduce nucleotide binding (5,23,46,48,52,60).…”

The Evolution of the Conserved ATPase Domain (CAD): Reconstructing the History of an Ancient Protein Module

“…The ATPase motif contained within the CAD is referred to as a Walker type motif (Walker et al 1982), and was first described after a comparison of a number of different ATPase sequences. Upon X-ray crystallographic determination of ATPase structures (Fry et al 1986), the Walker motif was shown to consist of three structural components: (1) a hydrophobic strand of parallel ␤-pleated sheet ending in an aspartate, (2) an ␣-helix of two lysines separated by three residues, two of which are hydrophobic, and (3) a glycine-rich flexible ''P-loop'' containing the highly conserved ''GKT'' sequence. The lysine residue in this motif is the only amino acid within the loop that is thought to directly contact bound Mg.ATP.…”