Conformation of Leu-Arg-Arg-Ala-Ser-Leu-Gly bound in the active site of adenosine cyclic 3',5'-phosphate dependent protein kinase

Hn, Bramson; Ne, Thomas; Wt, Miller; Dc, Fry; As, Mildvan; Et, Kaiser

doi:10.1021/bi00388a042

Cited by 22 publications

(9 citation statements)

References 10 publications

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“…[18][19][20] The 20-residue inhibitory Alapeptide, containing the sequence -RRNAI-is an analog of the substrate peptide that contains the sequence -RRASI-and that can be phosphorylated on serine. Such peptides assume an extended conformation at the active site of the enzyme as found by NMR 22,23 and by X-ray diffraction. 19,24 As found by NMR with parallel kinetic assays, in solution the enzyme-bound ATP shows a high-antiglycosyl torsional angle ( ϭ 78 Ϯ 10°) when the enzyme is fully active, which decreases to a low-antiglycosyl torsional angle ( ϳ30°) as activity is lost.…”

Section: Protein Kinasesmentioning

confidence: 78%

Mechanisms of signaling and related enzymes

Mildvan¹

1997

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Most enzymes involved in cell signaling, such as protein kinases, protein phosphatases, GTPases, and nucleotide cyclases catalyze nucleophilic substitutions at phosphorus. When possible, the mechanisms of such enzymes are most clearly described quantitatively in terms of how associative or dissociative they are. The mechanisms of cell signaling enzymes range from < or = 8% associative (cAMP-dependent protein kinase) to approximately 50% associative (G protein Gi alpha 1). Their catalytic powers range from 10(5.7) (p21ras) to 10(11.7) (lambda Ser-Thr protein phosphatase), usually comparable in magnitude with those of nonsignaling enzymes of the same mechanistic class. Exceptions are G proteins, which are 10(3)- to 10(5)-fold poorer catalysts than F1 and myosin ATPases. The lower catalytic powers of G proteins may be ascribed to the absence of general base catalysis, and additionally in the case of p21ras, to the absence of a catalytic Arg residue, which interacts with the transition state. From kinetic studies of mutant and metal ion substituted enzymes, the catalytic powers of cell signaling and related enzymes can be rationalized quantitatively by factors contributed by metal ion catalysis (> or = 10(5), general acid catalysis (approximately 10(3 +/- 1)), general base catalysis (approximately 10(3 +/- 1)), and transition-state stabilization by cationic and hydrogen bond donating residues (approximately 10(3 +/- 1)).

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Section: Protein Kinasesmentioning

confidence: 78%

Mechanisms of signaling and related enzymes

Mildvan¹

1997

Proteins

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256

274

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“…Eight different ADR1 peptide analogs were synthesized, each containing an amino acid change corresponding to an ADRJC mutation, and the ability of each peptide to be phosphorylated by yeast cAPK was analyzed. Some ADRJC mutations causing alterations in previously known important recognition determinants of cAPK (e.g., R227L, R228K, F231S, and S232R) (4,24) had very dramatic effects on cAPK phosphorylation of Ser-230. These alterations increased the Km for phosphorylation by 20-to 400-fold (Table 4).…”

Section: Resultsmentioning

confidence: 99%

ADR1c mutations enhance the ability of ADR1 to activate transcription by a mechanism that is independent of effects on cyclic AMP-dependent protein kinase phosphorylation of Ser-230.

et al. 1992

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Four ADR1c mutations that occur close to Ser-230 of the Saccharomyces cerevisiae transcriptional activator ADR1 and which greatly enhance the ability of ADR1 to activate ADH2 expression under glucose-repressed conditions have been shown to reduce or eliminate cyclic AMP-dependent protein kinase (cAPK) phosphorylation of Ser-230 in vitro. In addition, unregulated cAPK expression in vivo blocks ADH2 depression in an ADR1-dependent fashion in which ADR1c mutations display decreased sensitivity to unregulated cAPK activity. Taken together, these data have suggested that ADR1c mutations enhance ADR1 activity by blocking cAPK phosphorylation and inactivation of Ser-230. We have isolated and characterized an additional 17 ADR1c mutations, defining 10 different amino acid changes, that were located in the region defined by amino acids 227 through 239 of ADR1. Three observations, however, indicate that the ADR1c phenotype is not simply equivalent to a lack of cAPK phosphorylation. First, only some of these newly isolated ADR1c mutations affected the ability of yeast cAPK to phosphorylate corresponding synthetic peptides modeled on the 222 to 234 region of ADR1 in vitro. Second, we observed that strains lacking cAPK activity did not display enhanced ADH2 expression under glucose growth conditions. Third, when Ser-230 was mutated to a nonphosphorylatable residue, lack of cAPK activity led to a substantial increase in ADH2 expression under glucose-repressed conditions. Thus, while cAPK controls ADH2 expression and ADR1 is required for this control, cAPK acts by a mechanism that is independent of effects on ADR1 Ser-230. It was also observed that deletion of the ADR1c region resulted in an ADR1c phenotype. The ADR1c region is, therefore, involved in maintaining ADR1 in an inactive form. ADR1c mutations may block the binding of a repressor to ADR1 or alter the structure of ADR1 so that transcriptional activation regions become unmasked.

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“…In addition to the highly conserved bZIP region, ACR1 contains the sequence KRRMS (residues 376 to 380), which conforms to both consensus sequences (KRRXS and RRXS) for phosphorylation by cAMP-dependent protein kinases (3,28). Otherwise, the putative structural gene for ACR1 does not contain any other recognizable sequence motifs.…”

Section: Methodsmentioning

confidence: 99%