Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.11.11 (AMPK)
12,425 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The details of the process by which protein kinase catalyzes phosphoryl group transfers are beginning to be understood. Early work that explored the primary specificity of cAMP-dependent protein kinase action enabled the synthesis of small peptide substrates for the enzyme. Enzyme-peptide interactions seem simpler to understand than protein-protein interactions, so peptide substrates have been used in most protein kinase studies. In most investigations the kinetics for the phosphorylation of small peptides have been interpreted as being consistent with mechanisms which do not invoke phospho-enzyme intermediates (see, for example, Bolen et al.). Protein kinase has been shown to bind two metal ions in the presence of a nucleotide. Using magnetic resonance techniques the binding of these ions has been utilized to elucidate the conformation of nucleotide and peptide substrates or inhibitors when bound in the enzymic active site. Also, two new peptides with the form Leu-Arg-Arg-Ala-Ser-Y-Gly, where Y was either Pro or (N-methyl)Leu, were synthesized and found not to be substrates, within the limits of detection, for protein kinase. The striking lack of affinity that protein kinase has for such peptides which are unlikely to form a beta 3-6 turn has not been reported before. Our results may indicate that this type of turn is a requirement for protein kinase catalyzed phosphorylation or that these peptides lack the ability to form a particular hydrogen bond with the enzyme. Magnetic resonance techniques have indicated that the distance between the phosphorous in the gamma-phosphoryl group of MgATP and the hydroxyl oxygen of serine in the peptide Leu-Arg-Arg-Ala-Ser-Leu-Gly is 5.3 +/- 0.7 A. This, together with certain kinetic evidence, suggests that the mechanism by which protein kinase catalyzes phosphoryl group transfer has considerable dissociative character. Chemical modifications, including one using a peptide-based affinity label, have identified two residues at or near the active site, lysine-72 and cysteine 199. While neither of these groups has been shown to be catalytically essential, similar studies may help to identify groups that are directly involved in the catalytic process. Finally, a spectrophotometric assay for cAMP-dependent protein kinase has been described. Using this assay the preliminary results of an in-depth study of the pH dependence of protein kinase catalyzed phosphoryl group transfer have been obtained. This study shall aid in the identification of active site residues and should contribute to the elucidation of the enzyme's catalytic mechanism.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Mechanistic studies of cAMP-dependent protein kinase action. 636 50

A series of synthetic peptides corresponding to the amino-terminal region of chicken gizzard myosin light chain (Mr 20 000) have been tested for their capacity to act as substrates for the cAMP-dependent protein kinase. The 18-residue peptide, K6AKTTK11 K12R13PQRATS19NVFS , was stoichiometrically phosphorylated on serine-19 by the cAMP-dependent protein kinase. This is the same residue phosphorylated by the myosin light chain kinase. The cAMP-dependent protein kinase phosphorylated this peptide with an apparent Km of 120 microM and Vmax of 0.29 mumol . min .-1 mg-1. The Km is 17-fold higher and the Vmax 10-fold lower than the corresponding values obtained with this peptide as substrate for the myosin light chain kinase. The kinetics of phosphorylation of shortened peptides corresponding to this 18-residue sequence together with those of another related sequence, RPQRAKAKTTKATSNVFS , indicated that the myosin light chain kinase had a relatively stronger dependence on lysine residues, whereas the cAMP-dependent protein kinase depends more on arginine residues. Although both the cAMP-dependent protein kinase and the myosin light chain kinase phosphorylate the same serine in the myosin light chain peptides, these enzymes are influenced by different nearby basic residues.
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PMID:Comparison of substrate specificity of myosin kinase and cyclic AMP-dependent protein kinase. 654 60

Rat liver ribosomes and 40 S ribosomal subunits were phosphorylated with the purified catalytic subunit of cAMP-dependent protein kinase. Phosphorylation of ribosomal protein S6 plateaued at around 2 mol of phosphate/mol of protein with both substrates. Peptide map analyses showed that the most prominent phosphorylation sites associated with 40 S substrates were the adjacent serines in the Arg-Leu-Ser-Ser-Leu-Arg segment of S6. The first serine residue appeared to be the preferred site as has been established previously for 80 S ribosomes (Wettenhall, R.E.H., and Cohen, P. (1982) FEBS Lett. 140, 263-269). Additional phosphorylation sites were apparent from the peptide maps. One of these was associated with the triphosphopeptide (termed T1a) having the sequence Arg-Leu-Ser-Ser-Leu-Arg-Ala-Ser-Thr-Ser-Lys. A larger fragment of S6 (termed Tlc) isolated from mild tryptic digests of whole ribosomes, consisted of the T1a sequence extended by the sequence Ser-Glu-Glu-Ser-Gln-(Lys) at the COOH terminus. A comparison of the size and chromatographic and isoelectric focusing properties of the T1a/T1c peptides and prominent tryptic peptides of S6 from insulin-stimulated hepatocytes indicated a relationship between these peptides. Thus, it appeared that some of the potential phosphorylation sites in the T1a/T1c region of S6 are phosphorylated by an insulin-regulated kinase in hepatocytes.
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PMID:Phosphorylation of hepatic ribosomal protein S6 on 80 and 40 S ribosomes. Primary structure of S6 in the region of the major phosphorylation sites for cAMP-dependent protein kinases. 669 58

A synthetic heptadecapeptide corresponding to part of the NH2-terminal 17 residues of chicken gizzard myosin light chain (Mr = 20,000), Ser-Ser-Lys-Thr-Thr-Lys-Arg-Pro-Gln-Arg-Ala-Thr-Ser-(P)-Asn-Val-Phe-Ser-NH2, was readily phosphorylated by the myosin light chain kinase isolated from the same tissue. The synthetic peptide was phosphorylated stoichiometrically at serine 13, the same residue phosphorylated in the parent protein. The apparent Km and Vmax for peptide phosphorylation was 90 microM and 1.3 mumol min-1 mg-1 compared to 10 microM and 22 mumol min-1 mg-1, respectively, for the myosin light chain. The synthetic heptadecapeptide acted as a competitive inhibitor for myosin light chain phosphorylation with Ki approximately 600 microM. Acetylation of the heptadecapeptide alpha-amino group of serine 1 had little effect on Vmax (0.8 mumol min-1 mg-1) and increased the apparent Km 2-fold. The smooth muscle myosin light chain kinase did not phosphorylate the synthetic heptadecapeptide analog of the corresponding skeletal muscle myosin light chain (Mr = 18,500), nor did it phosphorylate synthetic peptide substrates specific for the cAMP-dependent protein kinase or phosphorylase b kinase. These findings support the idea that the myosin light chain kinase has particular protein substrate specificity requirements and that some of these are derived from the region of primary structure around the phosphorylation site in its native substrate.
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PMID:Phosphorylation of a synthetic heptadecapeptide by smooth muscle myosin light chain kinase. 689 43

The substrate binding properties of skeletal muscle myosin light chain kinase were investigated with a synthetic peptide containing the photoreactive amino acid p-benzoylphenylalanine (Bpa) incorporated amino-terminal of the phosphoacceptor serine (BpaKKRAARATSNVFA). When photolyzed at 350 nm, the peptide was cross-linked stoichiometrically to myosin light chain kinase in a Ca2+/calmodulin-dependent manner. Peptide incorporation into kinase inhibited light chain phosphorylation, and the loss of kinase activity was proportional to the extent of peptide incorporated. After peptide I was incorporated into myosin light chain kinase, it was partially phosphorylated in the absence of Ca2+/calmodulin. The extent of phosphorylation increased in the presence of Ca2+/calmodulin. The cross-linked photoadduct was digested, labeled peptides were purified by high performance liquid chromatography, and sites of covalent modification were determined by amino acid sequencing and analysis. The covalent modification in the catalytic core occurred on Ile-373 (66%) and in a peptide containing residues Asn-422 to Met-437 (14%), respectively. Lys-572 in the autoinhibitory region accounted for 20% of the incorporated label. The coincident covalent modification of the autoinhibitory domain suggests that it is located near the catalytic site. Based upon a model of the catalytic core, the substrate peptide is predicted to bind in the cleft between the two lobes of the kinase. The orientation of the substrate peptide on myosin light chain kinase is similar to the orientation of the substrate recognition fragment, but not the high affinity binding fragment, of inhibitor peptide of cAMP-dependent protein kinase in the catalytic subunit of the cAMP-dependent protein kinase.
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PMID:Photoaffinity labeling of a peptide substrate to myosin light chain kinase. 773 Mar 16

It was previously proposed that the activation of rat liver phenylalanine hydroxylase (EC 1.14.16.1) by cAMP-dependent protein kinase-mediated phosphorylation of Ser-16 is due to the introduction of the negatively charged phosphate group. To explore the validity of this proposal, we have applied site-directed mutagenesis to specifically replace Ser-16 with negatively charged amino acids, glutamic and aspartic; with polar uncharged amino acids, asparagine and glutamine; with the positively charged amino acid lysine; and with the nonpolar hydrophobic amino acid alanine. The wild-type and mutant enzymes were purified to homogeneity, and the importance of Ser-16 in the activation of phenylalanine hydroxylase was examined by comparing the state of activation of the phosphorylated form of the wild-type hydroxylase with that of the mutants. The kinetic studies carried out on the wild-type phosphorylated hydroxylase showed that all the activation could be accounted for by an increase in Vmax with no change in Km for either phenylalanine or the pterin cofactor. Replacement of Ser-16 with a negatively charged residue, glutamate of aspartate, resulted in the activation of the hydroxylase by 2- to 4-fold, whereas replacement with glutamine, asparagine, lysine, or alanine resulted in a much more modest increase. Further, lysolecithin was found to stimulate the phosphorylated hydroxylase and the mutant enzymes S16E and S16D by a factor of 6-7. In contrast, the mutants S16Q, S16N, and S16A all showed the same magnitude of activation as the wild-type with lysolecithin. Therefore, this study demonstrates that activation of the enzyme by phosphorylation of Ser-16 by cAMP-dependent protein kinase is due to the introduction of negative charge(s) and strongly suggests the involvement of electrostatic interaction between the regulatory and catalytic domains of the hydroxylase.
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PMID:Further studies of the role of Ser-16 in the regulation of the activity of phenylalanine hydroxylase. 776 94

X-linked agammaglobulinemia (XLA) is a hereditary defect of B-cell differentiation in man caused by deficiency of Bruton tyrosine kinase (BTK). A three-dimensional model for the BTK kinase domain, based on the core structure of cAMP-dependent protein kinase, was used to interpret the structural basis for disease in eight independent point mutations in patients with XLA. As Arg-525 of BTK has been thought to functionally substitute for a critical lysine residue in protein-serine kinases, the mutation Arg-525-->Gln was studied and found to abrogate the tyrosine kinase activity of BTK. All of the eight mutations (Lys-430-->Glu, Arg-520-->Glu, Arg-525-->Gln, Arg-562-->Pro, Ala-582-->Val, Glu-589-->Gly, Gly-594-->Glu, and Gly-613-->Asp) were located on one face of the BTK kinase domain, indicating structural clustering of functionally important residues.
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PMID:Structural basis for chromosome X-linked agammaglobulinemia: a tyrosine kinase disease. 780 24

A vincristine-resistant lymphoma cell line (HOB1/VCR1.0) that is resistant to 1.0 microM of vincristine has been established from a human immunoblastic B lymphoma cell line, HOB1. HOB1/VCR1.0 cells demonstrated the typical multidrug resistant phenotypes. Using two-dimensional gel electrophoresis, we discovered one protein with a molecular mass of 22 kDa and pI 5.7 that was overexpressed in HOB1/VCR1.0 cells. This protein was purified to the degree of apparent homogeneity by preparative isoelectric focusing and sodium dodecylsulfate-polyacrylamide gel electrophoresis. The identification of this protein with sorcin was revealed by comparing the internal amino acid sequence of three Lys-C digested peptides from the purified protein with the sequence previously determined for hamster sorcin. The complete primary structure of the human sorcin was deduced from nucleotide sequence analysis of its cDNA clones. It is composed of 198 amino acid residues with a calculated molecular weight of 21,676, and its sequence is highly similar to that of hamster sorcin (95%). Direct-binding assay with calcium showed that human sorcin is a calcium-binding protein with four 'E-F hand' structures typical of calcium-binding sites. Like the sorcin of hamster, two of the calcium-binding sites of human sorcin contain putative recognition sites for cAMP-dependent protein kinase. Southern and Northern blot analyses showed that the human sorcin gene was greatly amplified and overexpressed in resistant HOB1/VCR1.0 cells but not detected in the parental HOB1 cells. The overproduction of this protein in resistant cells implies that sorcin plays a role in expression of the resistant phenotype.
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PMID:Isolation and molecular cloning of human sorcin a calcium-binding protein in vincristine-resistant HOB1 lymphoma cells. 787 2

Colligin is a collagen-binding glycoprotein localized to the endoplasmic reticulum (ER) and has been proposed to play a role in collagen biosynthesis. Its membership in the serpin family prompted us to examine its effect on procollagen degradation. We first showed that procollagen degradation can take place in the ER of L6 myoblasts by using brefeldin A to block transit from the ER. This degradation could be prevented by the serine protease inhibitors N-tosyl-L-lysine chloromethyl ketone (TLCK) and N-tosyl-L-phenylalanine chloromethyl ketone (TPCK). To examine procollagen degradation in vitro, isolated liver microsomes were incubated with procollagen. Intact microsomes were unable to degrade labeled procollagen I, fibronectin, or the cytoplasmic proteins, phosphorylase b and the RI subunit of the cAMP-dependent protein kinase. However, when the microsomes were permeabilized by treatment with detergent, they became capable of degrading procollagen and fibronectin, but not the cytoplasmic proteins. The degrading activity was not due to cross-contamination by lysosomal or cytoplasmic, multicatalytic proteases. The proteolysis of procollagen chains in the treated microsomes was partially inhibited by TPCK, TLCK, and leupeptin. The most effective inhibitor was, however, colligin. In its presence, the breakdown of procollagen I, but not of fibronectin, was specifically inhibited. Colligin itself was not degraded by the microsomal preparations. The protein degrading activity was localized to the microsomal membranes, and showed a pH optimum of about 8.0. From these studies it is inferred that one of the roles of colligin may be to protect the procollagen I chains in the ER from degradation prior to their transport to the cis-Golgi compartment.
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PMID:Inhibition of procollagen I degradation by colligin: a collagen-binding serpin. 794

The crystal structure of ternary and binary substrate complexes of the catalytic subunit of cAMP-dependent protein kinase has been refined at 2.2 and 2.25 A resolution, respectively. The ternary complex contains ADP and a 20-residue substrate peptide, whereas the binary complex contains the phosphorylated substrate peptide. These 2 structures were refined to crystallographic R-factors of 17.5 and 18.1%, respectively. In the ternary complex, the hydroxyl oxygen OG of the serine at the P-site is 2.7 A from the OD1 atom of Asp 166. This is the first crystallographic evidence showing the direct interaction of this invariant carboxylate with a peptide substrate, and supports the predicted role of Asp 166 as a catalytic base and as an agent to position the serine -OH for nucleophilic attack. A comparison of the substrate and inhibitor ternary complexes places the hydroxyl oxygen of the serine 2.7 A from the gamma-phosphate of ATP and supports a direct in-line mechanism for phosphotransfer. In the binary complex, the phosphate on the Ser interacts directly with the epsilon N of Lys 168, another conserved residue. In the ternary complex containing ATP and the inhibitor peptide, Lys 168 interacts electrostatically with the gamma-phosphate of ATP (Zheng J, Knighton DR, Ten Eyck LF, Karlsson R, Xuong NH, Taylor SS, Sowadski JM, 1993, Biochemistry 32:2154-2161). Thus, Lys 168 remains closely associated with the phosphate in both complexes. A comparison of this binary complex structure with the recently solved structure of the ternary complex containing ATP and inhibitor peptide also reveals that the phosphate atom traverses a distance of about 1.5 A following nucleophilic attack by serine and transfer to the peptide. No major conformational changes of active site residues are seen when the substrate and product complexes are compared, although the binary complex with the phosphopeptide reveals localized changes in conformation in the region corresponding to the glycine-rich loop. The high B-factors for this loop support the conclusion that this structural motif is a highly mobile segment of the protein.
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PMID:cAMP-dependent protein kinase: crystallographic insights into substrate recognition and phosphotransfer. 800 55


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