Biomaterial Database

The ATP binding site on rho protein. Affinity labeling of Lys181 by pyridoxal 5'-diphospho-5'-adenosine. 1988

A J Dombroski, and J R LaDine, and R L Cross, and T Platt

Department of Biochemistry, University of Rochester Medical Center, New York 14642.

We have labeled the nucleoside triphosphate-binding domain of Escherichia coli rho factor with the ATP affinity analog [3H]pyridoxal 5'-diphospho-5'-adenosine (PLP-AMP). PLP-AMP completely inactivates the RNA-dependent ATPase activity of rho upon incorporation of 3 mol of reagent/mol of hexameric rho protein. Although the potency of PLP-AMP is enhanced when an RNA substrate such as poly(C) is present, the stoichiometry for inhibition remains the same as in the absence of poly(C). The nucleotide substrate ATP competes very effectively for the binding site and protects against PLP-AMP inactivation. A domain of rho called N2, which comprises the distal two-thirds of the molecule (residues 152-419) and encompasses the region proposed to bind ATP, is labeled specifically in the presence of poly(C). Amino acid sequence analysis of the single [3H]PLP-AMP labeled proteolytic fragment showed Lys181 to be the site of modification, suggesting that this residue normally interacts with the gamma-phosphoryl of bound ATP. These results agree with our proposed tertiary structure for the ATP-binding domain of rho that places this lysine residue in a flexible loop above a hydrophobic nucleotide-binding pocket comprised of several parallel beta-strands, similar to adenylate kinase, F1-ATPase, and related ATP-binding proteins. Parallel studies of rho structure and function by site-directed mutagenesis and chemical modification support this interpretation.

UI	MeSH Term	Description	Entries
D008239	Lysine	An essential amino acid. It is often added to animal feed.	Enisyl,L-Lysine,Lysine Acetate,Lysine Hydrochloride,Acetate, Lysine,L Lysine
D008969	Molecular Sequence Data	Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories.	Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D011066	Poly C	A group of cytosine ribonucleotides in which the phosphate residues of each cytosine ribonucleotide act as bridges in forming diester linkages between the ribose moieties.	Cytosine Polynucleotides,Polycytidylic Acid,Polycytidylic Acids,Acid, Polycytidylic,Acids, Polycytidylic,Polynucleotides, Cytosine
D011732	Pyridoxal Phosphate	This is the active form of VITAMIN B 6 serving as a coenzyme for synthesis of amino acids, neurotransmitters (serotonin, norepinephrine), sphingolipids, aminolevulinic acid. During transamination of amino acids, pyridoxal phosphate is transiently converted into pyridoxamine phosphate (PYRIDOXAMINE).	Pyridoxal 5-Phosphate,Pyridoxal-P,Phosphate, Pyridoxal,Pyridoxal 5 Phosphate,Pyridoxal P
D002851	Chromatography, High Pressure Liquid	Liquid chromatographic techniques which feature high inlet pressures, high sensitivity, and high speed.	Chromatography, High Performance Liquid,Chromatography, High Speed Liquid,Chromatography, Liquid, High Pressure,HPLC,High Performance Liquid Chromatography,High-Performance Liquid Chromatography,UPLC,Ultra Performance Liquid Chromatography,Chromatography, High-Performance Liquid,High-Performance Liquid Chromatographies,Liquid Chromatography, High-Performance
D000244	Adenosine Diphosphate	Adenosine 5'-(trihydrogen diphosphate). An adenine nucleotide containing two phosphate groups esterified to the sugar moiety at the 5'-position.	ADP,Adenosine Pyrophosphate,Magnesium ADP,MgADP,Adenosine 5'-Pyrophosphate,5'-Pyrophosphate, Adenosine,ADP, Magnesium,Adenosine 5' Pyrophosphate,Diphosphate, Adenosine,Pyrophosphate, Adenosine
D000255	Adenosine Triphosphate	An adenine nucleotide containing three phosphate groups esterified to the sugar moiety. In addition to its crucial roles in metabolism adenosine triphosphate is a neurotransmitter.	ATP,Adenosine Triphosphate, Calcium Salt,Adenosine Triphosphate, Chromium Salt,Adenosine Triphosphate, Magnesium Salt,Adenosine Triphosphate, Manganese Salt,Adenylpyrophosphate,CaATP,CrATP,Manganese Adenosine Triphosphate,MgATP,MnATP,ATP-MgCl2,Adenosine Triphosphate, Chromium Ammonium Salt,Adenosine Triphosphate, Magnesium Chloride,Atriphos,Chromium Adenosine Triphosphate,Cr(H2O)4 ATP,Magnesium Adenosine Triphosphate,Striadyne,ATP MgCl2
D000345	Affinity Labels	Analogs of those substrates or compounds which bind naturally at the active sites of proteins, enzymes, antibodies, steroids, or physiological receptors. These analogs form a stable covalent bond at the binding site, thereby acting as inhibitors of the proteins or steroids.	Affinity Labeling Reagents,Labeling Reagents, Affinity,Labels, Affinity,Reagents, Affinity Labeling
D000595	Amino Acid Sequence	The order of amino acids as they occur in a polypeptide chain. This is referred to as the primary structure of proteins. It is of fundamental importance in determining PROTEIN CONFORMATION.	Protein Structure, Primary,Amino Acid Sequences,Sequence, Amino Acid,Sequences, Amino Acid,Primary Protein Structure,Primary Protein Structures,Protein Structures, Primary,Structure, Primary Protein,Structures, Primary Protein
D001665	Binding Sites	The parts of a macromolecule that directly participate in its specific combination with another molecule.	Combining Site,Binding Site,Combining Sites,Site, Binding,Site, Combining,Sites, Binding,Sites, Combining