Pre-Steady-State Kinetic Analysis of cAMP-Dependent Protein Kinase Using Rapid Quench Flow Techniques

Grant, Bruce D.; Adams, Joseph A.

doi:10.1021/bi952144+

Cited by 121 publications

(252 citation statements)

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“…Because the rC[E230Q] data fit both Equation 2 as well as a linear function, a burst rate constant is not reported. These fits both yielded a well-defined linear rate constant of 6.0 f 0.1 s-', which was corrected to 15 s" (1.5-fold perturbed with respect to wild-type C-subunit) based on the wild-type C-subunit linear rate constant's hyperbolic dependence on [Kemptide] (Grant & Adams, 1996). The nearly linear plot for rC [E230Q] clearly indicates that the mutation decreases the phosphoryl transfer rate constant, causing it to become partially rate limiting.…”

Section: Pre-steady-state Kinetic Analysis Of Rc[e230qlmentioning

confidence: 99%

“…The apparatus contains three syringes driven by a stepping motor. Quench-flow experiments were executed as described previously (Grant & Adams, 1996).…”

Section: Rapid Quench-flow Measurementsmentioning

confidence: 99%

“…Stereochemical ( H o et at., 1988) and isotope partitioning (Kong & Cook, 1988) analyses of the reaction products indicated that phosphoryl transfer proceeds through a one-step, essentially irreversible process. Pre-steady-state quench-flow (Grant & Adams, 1996) kinetic studies measured phosphoryl transfer and product release rate constants of 500 and 20 s", respectively. Quenchflow kinetic analysis also equated the directly measured Kd for the substrate peptide Kemptide (LRRASLG), with the Ki for an inhibitor analogue (LRRAALG).…”

mentioning

confidence: 99%

“…In this study, the wild-type C-subunit kinetic reaction pathway is compared with kinetic reaction pathway for rC [E230Q]. (Yoon & Cook, 1987) for wild-type C-subunit, and K;(LRRAALG) equals the K,(Kemptide) (Grant & Adams, 1996). A characteristic y-axis intersection point in a double-reciprocal initial velocity versus [Kemptide] plot (with inhibitor concentration as a fixed variable) established that LRRAALG is a competitive inhibitor with respect to Kemptide in rC[E230Q] (data not shown).…”

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confidence: 99%

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Examination of an active‐site electrostatic node in the cAMP‐dependent protein kinase catalytic subunit

et al. 1996

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The active site of the CAMP-dependent protein kinase catalytic subunit harbors a cluster of acidic residuesAsp 127, Glu 170, Glu 203, Glu 230, and Asp 241 -that are not conserved throughout the protein kinase family. Based on crystal structures of the catalytic subunit, these amino acids are removed from the site of phosphoryl transfer and are implicated in substrate recognition. Glu 230, the most buried of these acidic residues, was mutated to Ala (rC[E230A]) and Gln (rC[E230Q]) and overexpressed in Escherichia coli. In contrast to the mostly insoluble and destabilized rC[E230A], rC[E230Q] is largely soluble, purifies like wild-type enzyme, and displays wild-type-like thermal stability. The mutation in rC[E230Q] causes an order of magnitude decrease in the affinity for a heptapeptide substrate, Kemptide. In addition, two independent kinetic techniques were used to dissect phosphoryl transfer and product release steps in the reaction pathway. Viscosometric and pre-steady-state quench-flow analyses revealed that the phosphoryl transfer rate constant decreases by an order of magnitude, whereas the product release rate constant remains unperturbed. Electrostatic alterations in the rC[E230Q] active site were assessed using modeling techniques that provide molecular interpretations for the substrate affinity and phosphoryl transfer rate decreases observed experimentally. These observations indicate that subsite recognition elements in the catalytic subunit make electrostatic contributions that are important not only for peptide affinity, but also for catalysis. Protein kinases may, therefore, discriminate substrates by not only binding them tightly, but also by only turning over ones that complement the electrostatic character of the active site.Keywords: CAMP-dependent protein kinase; catalytic subunit; electrostatic interaction; pre-steady-state kinetics; substrate specificityThe catalytic subunit of CAMP-dependent protein kinase is the simplest member of the protein kinase family because it is one of the smallest and its activity is regulated by a separate subunit (R-subunit). The holoenzyme form of the enzyme is an inactive complex composed of an R-subunit homodimer and two C-subunits (Taylor et al., 1989). Active monomeric C-subunits are released after CAMP binds with high affinity to the regulatory subunits (Gill & Garren, 1969) and phosphorylate a broad range of protein substrates involved in metabolic (Krebs, 1985), transcriptional (Yamamoto et al., 1988), and signal transduction events (Cook & McCormick, 1993). Active C-subunit phosphor- ylates protein substrates on Ser and Thr residing in a common consensus sequence, Arg-Arg-X-Ser/Thr-Y, where X denotes any amino acid and Y is a hydrophobic residue (Zetterqvist et al., 1990). The P -3 and P -2 arginines3 confer 4-6 kcal/ mol of binding energy (Walsh et al., 1990) to a 20-amino acid C-subunit inhibitor (IP20). C-subunit amino acids that complement these arginines were first visualized in a crystal structure of the C-subunit complexed with IP20 (Knigh...

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Section: Pre-steady-state Kinetic Analysis Of Rc[e230qlmentioning

confidence: 99%

“…The apparatus contains three syringes driven by a stepping motor. Quench-flow experiments were executed as described previously (Grant & Adams, 1996).…”

Section: Rapid Quench-flow Measurementsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Examination of an active‐site electrostatic node in the cAMP‐dependent protein kinase catalytic subunit

et al. 1996

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“…The data are interpreted according to the wild-type kinetic mechanism. This mechanism, shown in Scheme 1, is derived from a combination of rapid quench flow and viscosometric studies (25,26).…”

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Kinetic Analyses of Mutations in the Glycine-Rich Loop of cAMP-Dependent Protein Kinase

et al. 1998

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The conserved glycines in the glycine-rich loop (Leu-Gly 50 -Thr-Gly 52 -Ser-Phe-Gly 55 -ArgVal) of the catalytic (C) subunit of cAMP-dependent protein kinase were each mutated to Ser (G50S, G52S, and G55S). The effects of these mutations were assessed here using both steady-state and presteady-state kinetic methods. While G50S and G52S reduced the apparent affinity for ATP by approximately 10-fold, substitution at Gly55 had no effect on nucleotide binding. In contrast to ATP, only mutation at position 50 interfered with ADP binding. These three mutations lowered the rate of phosphoryl transfer by 7-300-fold. The combined data indicate that G50 and G52 are the most critical residues in the loop for catalysis, with replacement at position 52 being the most extreme owing to a larger decrease in the rate of phosphoryl transfer (29 vs 1.6 s -1 in contrast to 500 s -1 for wild-type C). Surprisingly, all three mutations lowered the affinity for Kemptide by approximately 10-fold, although none of the loop glycines makes direct contact with the substrate. The inability to correlate the rate constant for net product release with the dissociation constant for ADP implies that other steps may limit the decomposition of the ternary product complex. The observations that G52S (a) selectively affects ATP binding and (b) significantly lowers the rate of phosphoryl transfer without making direct contact with either the nucleotide or the peptide imply that this residue serves a structural role in the loop, most likely by positioning the backbone amide of Ser53 for contacting the γ-phosphate of ATP. Energyminimized models of the mutant proteins are consistent with the observed kinetic consequences of each mutation. The models predict that only mutation of Gly52 will interfere with the observed hydrogen bonding between the backbone and ATP.Many nucleotide binding enzymes utilize glycine-rich loops that help position nucleotides for catalysis (1-3). Crystal structures of dinucleotide and mononucleotide binding enzymes define several glycine-rich loops that are distinct both in their amino acid sequence and in their orientation relative to the nucleotide. The glycine-rich loops found in dinucleotide binding enzymes [e.g., pyruvate dehydrogenase (4) and dihydrofolate reductase (5)] are typically Gly-X-Gly-X-X-Gly and provide space for the dinucleotide pyrophosphate moiety. The glycine-rich loops, or P-loops, found in mononucleotide binding enzymes [e.g., p21ras (6), adenylate kinase (7), Rec A (8), and elongation factor Tu (9)] have the consensus sequence Gly-X-X-X-X-Gly-Lys-Ser/Thr and interact with the γ-phosphate of ATP (10). In contrast, the protein kinase family of enzymes contain a glycine-rich loop with a consensus sequence Gly-X-Gly-X-X-Gly-X-Val. On the basis of numerous crystal structures, this loop is part of a -hairpin that connects -strands 1 and 2 and serves as a nucleotide-positioning loop (NPL) 1 that interacts with all three phosphates of ATP (11)(12)(13)(14)(15). The glycines in the protein kinase NPL are ...

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Mechanisms of signaling and related enzymes

Mildvan¹

1997

Proteins

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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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Pre-Steady-State Kinetic Analysis of cAMP-Dependent Protein Kinase Using Rapid Quench Flow Techniques

Cited by 121 publications

References 32 publications

Examination of an active‐site electrostatic node in the cAMP‐dependent protein kinase catalytic subunit

Examination of an active‐site electrostatic node in the cAMP‐dependent protein kinase catalytic subunit

Kinetic Analyses of Mutations in the Glycine-Rich Loop of cAMP-Dependent Protein Kinase

Mechanisms of signaling and related enzymes

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