Biomaterial Database

Differential labeling of the catalytic subunit of cAMP-dependent protein kinase with acetic anhydride: substrate-induced conformational changes. 1989

J A Buechler, and T A Vedvick, and S S Taylor

Department of Chemistry, University of California, San Diego, La Jolla 92093.

In order to identify regions that are sensitive to substrate-induced perturbations, the catalytic subunit of cAMP-dependent protein kinase was differentially labeled with [3H]acetic anhydride. Treatment of the catalytic subunit with acetic anhydride in the absence of substrates led to the irreversible inhibition of activity, and MgATP protected against inactivation. After development of a purification protocol for the lysine-containing peptides, the reactivity of each lysine in the native enzyme was calculated. The reactivity profile of lysines in the apoenzyme revealed three distinct regions. In general, the lysines within the amino-terminal segment (residues 1-83) and the carboxy-terminal segment (192-345) were relatively reactive. In contrast, the five lysines in the middle of the protein (Lys-92, -105, -111, -168, and -189) were very unreactive, indicating that these groups are sequestered from the aqueous solvent. The reactivity of each lysine was then determined in the presence of MgATP and in the presence of MgATP and a 20-residue inhibitor peptide. Most of the substrate-induced changes in lysine reactivity were localized in the amino-terminal segment, while the reactivities of lysines in the carboxy-terminal region were not altered significantly by MgATP or inhibitor peptide. MgATP affords substantial protection to three residues in particular. Lys-72, predicted previously to be essential for nucleotide binding was relatively reactive in the apoenzyme, whereas labeling was nearly abolished in the presence of MgATP.(ABSTRACT TRUNCATED AT 250 WORDS)

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
D008958	Models, Molecular	Models used experimentally or theoretically to study molecular shape, electronic properties, or interactions; includes analogous molecules, computer-generated graphics, and mechanical structures.	Molecular Models,Model, Molecular,Molecular Model
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
D009206	Myocardium	The muscle tissue of the HEART. It is composed of striated, involuntary muscle cells (MYOCYTES, CARDIAC) connected to form the contractile pump to generate blood flow.	Muscle, Cardiac,Muscle, Heart,Cardiac Muscle,Myocardia,Cardiac Muscles,Heart Muscle,Heart Muscles,Muscles, Cardiac,Muscles, Heart
D010446	Peptide Fragments	Partial proteins formed by partial hydrolysis of complete proteins or generated through PROTEIN ENGINEERING techniques.	Peptide Fragment,Fragment, Peptide,Fragments, Peptide
D011485	Protein Binding	The process in which substances, either endogenous or exogenous, bind to proteins, peptides, enzymes, protein precursors, or allied compounds. Specific protein-binding measures are often used as assays in diagnostic assessments.	Plasma Protein Binding Capacity,Binding, Protein
D011487	Protein Conformation	The characteristic 3-dimensional shape of a protein, including the secondary, supersecondary (motifs), tertiary (domains) and quaternary structure of the peptide chain. PROTEIN STRUCTURE, QUATERNARY describes the conformation assumed by multimeric proteins (aggregates of more than one polypeptide chain).	Conformation, Protein,Conformations, Protein,Protein Conformations
D011494	Protein Kinases	A family of enzymes that catalyze the conversion of ATP and a protein to ADP and a phosphoprotein.	Protein Kinase,Kinase, Protein,Kinases, Protein
D000085	Acetates	Derivatives of ACETIC ACID. Included under this heading are a broad variety of acid forms, salts, esters, and amides that contain the carboxymethane structure.	Acetate,Acetic Acid Esters,Acetic Acids,Acids, Acetic,Esters, Acetic Acid
D000089	Acetic Anhydrides	Compounds used extensively as acetylation, oxidation and dehydrating agents and in the modification of proteins and enzymes.	Anhydrides, Acetic