The Kinase Domain Alters the Kinetic Properties of the Myosin IIIA Motor†

Biochemistry ◽  
2008 ◽  
Vol 47 (8) ◽  
pp. 2485-2496 ◽  
Author(s):  
Andréa C. Dosé ◽  
Shobana Ananthanarayanan ◽  
Judy E. Moore ◽  
Amoreena C. Corsa ◽  
Beth Burnside ◽  
...  
Keyword(s):  
Kinase Domain ◽  
Kinetic Properties ◽  
Myosin Iiia

scholarly journals Mutagenesis of charged residues in a conserved sequence in the 2-kinase domain of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase

1997 ◽  
Vol 321 (3) ◽  
pp. 609-614 ◽  
Author(s):  
Luc BERTRAND ◽  
Didier VERTOMMEN ◽  
Ernest FEYTMANS ◽  
Attilio Di PIETRO ◽  
Mark H. RIDER ◽  
...  
Keyword(s):  
Structural Changes ◽  
Kinase Activity ◽  
Kinase Domain ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Kinetic Properties ◽  
Magnesium Citrate ◽  
Phosphate Binding ◽  
Conserved Sequence ◽  
Kinase Mutation

Arg-136, Glu-137, Arg-138 and Arg-139 are conserved in all sequences of the 2-kinase domain of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Their role was studied by site-directed mutagenesis. All the mutations had little, if any, effect on fructose-2,6-bisphosphatase activity. Mutations of Arg-136 and Glu-137 into Ala caused only minor modifications of phosphofructo-2-kinase activity. In contrast, mutation of Arg-138 into Ala increased 280-fold the Km for fructose 6-phosphate of phosphofructo-2-kinase. Mutation of Arg-139 into Ala resulted in decreases in phosphofructo-2-kinase Vmax/Km for MgATP and fructose 6-phosphate 600-fold and 5000-fold respectively. Mutation of Arg-139 into Lys and Gln increased the Km of phosphofructo-2-kinase for MgATP (20-fold and 25-fold respectively) and for fructose 6-phosphate (8-fold and 13-fold), and the IC50 for MgADP (30-fold and 50-fold) and for magnesium citrate (7-fold and 25-fold). However, these two mutations did not affect nucleotide binding, as measured by quenching of intrinsic fluorescence. The changes in kinetic properties induced by mutations could not be attributed to structural changes. It is proposed that Arg-138 is involved in fructose 6-phosphate binding and that Arg-139 is probably involved in the stabilization of the transition state and so participates in catalysis.

scholarly journals Crystal Structure of G Protein-coupled Receptor Kinase 5 in Complex with a Rationally Designed Inhibitor

2015 ◽  
Vol 290 (34) ◽  
pp. 20649-20659 ◽  
Author(s):  
Kristoff T. Homan ◽  
Helen V. Waldschmidt ◽  
Alisa Glukhova ◽  
Alessandro Cannavo ◽  
Jianliang Song ◽  
...  
Keyword(s):  
Crystal Structure ◽  
G Protein ◽  
Active Site ◽  
Parent Compound ◽  
Kinase Domain ◽  
Kinetic Properties ◽  
G Protein Coupled Receptor ◽  
Structure Based Drug Design ◽  
Terminal Structure ◽  
G Protein Coupled

G protein-coupled receptor kinases (GRKs) regulate cell signaling by initiating the desensitization of active G protein-coupled receptors. The two most widely expressed GRKs (GRK2 and GRK5) play a role in cardiovascular disease and thus represent important targets for the development of novel therapeutic drugs. In the course of a GRK2 structure-based drug design campaign, one inhibitor (CCG215022) exhibited nanomolar IC50 values against both GRK2 and GRK5 and good selectivity against other closely related kinases such as GRK1 and PKA. Treatment of murine cardiomyocytes with CCG215022 resulted in significantly increased contractility at 20-fold lower concentrations than paroxetine, an inhibitor with more modest selectivity for GRK2. A 2.4 Å crystal structure of the GRK5·CCG215022 complex was determined and revealed that the inhibitor binds in the active site similarly to its parent compound GSK180736A. As designed, its 2-pyridylmethyl amide side chain occupies the hydrophobic subsite of the active site where it forms three additional hydrogen bonds, including one with the catalytic lysine. The overall conformation of the GRK5 kinase domain is similar to that of a previously determined structure of GRK6 in what is proposed to be its active state, but the C-terminal region of the enzyme adopts a distinct conformation. The kinetic properties of site-directed mutants in this region are consistent with the hypothesis that this novel C-terminal structure is representative of the membrane-bound conformation of the enzyme.

scholarly journals Multiple Activation Loop Conformations and Their Regulatory Properties in the Insulin Receptor's Kinase Domain

2001 ◽  
Vol 276 (50) ◽  
pp. 46933-46940 ◽  
Author(s):  
Ararat J. Ablooglu ◽  
Mark Frankel ◽  
Elena Rusinova ◽  
John B. Alexander Ross ◽  
Ronald A. Kohanski
Keyword(s):  
Active Site ◽  
Catalytic Efficiency ◽  
Kinase Domain ◽  
Kinetic Properties ◽  
Activation Loop ◽  
Steady State Kinetic ◽  
Tryptic Cleavage ◽  
Loop Conformations ◽  
Steric Accessibility ◽  
Regulatory Properties

Low catalytic efficiency of protein kinases often results from intrasteric inhibition caused by the activation loop blocking the active site. In the insulin receptor's kinase domain, Asp-1161 and Tyr-1162 in the peptide substrate-like sequence of the unphosphorylated activation loop can interact with four invariant residues in the active site: Lys-1085, Asp-1132, Arg-1136, and Gln-1208. Contributions of these six residues to intrasteric inhibition were tested by mutagenesis, and the unphosphorylated kinase domains were characterized. The mutations Q1208S, K1085N, and Y1162F each relieved intrasteric inhibition, increasing catalytic efficiency but without changing the rate-limiting step of the reaction. The mutants R1136Q and D1132N were virtually inactive. Steric accessibility of the active site was ranked by relative changes in iodide quenching of intrinsic fluorescence, and A-loop conformation was ranked by limited tryptic cleavage. Together these ranked the openness of the active site cleft as R1136Q ≈ D1132N ≥ D1161A > Y1162F ≈ K1085N > Q1208S ≥ wild-type. These findings demonstrate the importance of specific invariant residues for intrasteric inhibition and show that diverse activation loop conformations can produce similar steady-state kinetic properties. This suggests a broader range of regulatory properties for the activation loop than expected from a simple off-versus-on switch for kinase activation.

scholarly journals Phosphorylation of the Kinase Domain Regulates Autophosphorylation of Myosin IIIA and Its Translocation in Microvilli

Biochemistry ◽  
2014 ◽  
Vol 53 (49) ◽  
pp. 7835-7845 ◽  
Author(s):  
Byung Chull An ◽  
Tsuyoshi Sakai ◽  
Shigeru Komaba ◽  
Hiroko Kishi ◽  
Sei Kobayashi ◽  
...  
Keyword(s):  
Kinase Domain ◽  
Myosin Iiia

scholarly journals Study of the roles of Arg-104 and Arg-225 in the 2-kinase domain of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase by site-directed mutagenesis

1995 ◽  
Vol 309 (1) ◽  
pp. 341-346 ◽  
Author(s):  
M H Rider ◽  
K M Crepin ◽  
M De Cloedt ◽  
L Bertrand ◽  
D Vertommen ◽  
...  
Keyword(s):  
Structural Changes ◽  
Kinase Domain ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Kinetic Properties ◽  
Wild Type ◽  
Guanidinium Chloride ◽  
Wild Type Enzyme ◽  
Ph Profile ◽  
Substrate Concentrations

The roles of Arg-104 and Arg-225 located in the 2-kinase domain of the bifunctional enzyme 6-phosphofructo-2-kinase (PFK-2)/fructose-2,6-bisphosphatase (FBPase-2) have been studied by site-directed mutagenesis. In recombinant rat liver PFK-2/FBPase-2, mutation of Arg-225 to Ser increased the Km of PFK-2 for fructose-6-phosphate (Fru-6-P) 7-fold at pH 6 and decreased PFK-2 activity at suboptimal substrate concentrations between pH 6 and 9.5. The mutation had no effect on the Vmax of PFK-2 or on the Km of PFK-2 for MgATP. The mutation also increased the Vmax. of FBPase-2 4-fold without changing the Km for Fru-2,6-P2 or IC50 of Fru-6-P. These findings are in agreement with a previous study [Rider and Hue (1992) Eur. J. Biochem. 207, 967-972] on the protection by Fru-6-P of the labelling of Arg-225 by phenylglyoxal, and suggest that Arg-225 participates in Fru-6-P binding. In recombinant rat muscle PFK-2/FBPase-2, mutation of Arg-104 to Ser increased the Km for Fru-6-P 60-fold, increased the IC50 of citrate, increased the Vmax. 1.5-3-fold at pH 8.5 and altered the pH profile of PFK-2 activity. It did not affect the Km of PFK-2 for MgATP. The mutation also decreased the Vmax. of FBPase-2 3-fold, increased the Km for Fru-2,6-P2 70-fold and increased the IC50 of Fru-6-P at least 300-fold. Although the dimeric structure was maintained in the mutant, its PFK-2 activity was more sensitive towards inactivation by guanidinium chloride than the wild-type enzyme activity. The findings indicate that Arg-104 is involved in Fru-6-P binding in the PFK-2 domain and that it might also bind citrate. Structural changes resulting from the mutation might be responsible for the changes in kinetic properties of FBPase-2.

Kinase Domain Mutants of Bcr-Abl Exhibit Altered Transformation Potency, Kinase Activity, and Substrate Utilization, Irrespective of Sensitivity to Imatinib.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4796-4796
Author(s):  
M. MacPartlin ◽  
T. O’Hare ◽  
T. Bumm ◽  
V. Goss ◽  
K. Lee ◽  
...  
Keyword(s):  
Signaling Pathways ◽  
Kinase Activity ◽  
Kinase Domain ◽  
Full Length ◽  
Kinase Assay ◽  
In Vitro Assays ◽  
Kinetic Properties ◽  
Resistance Mutations ◽  
Differential Activation

Abstract Background: Kinase domain (KD) mutations of Bcr-Abl interfering with imatinib binding are the major mechanism of acquired resistance. Mutations of the ATP binding p-loop are associated with a poor prognosis compared to other mutations, irrespective of imatinib sensitivity. To elucidate the underlying mechanisms we compared five KD mutants in biological and biochemical assays. Methods and Results: In competition experiments using Ba/F3 cells expressing native or KD mutated Bcr-Abl, proliferation rates of Y253F and E255K were highest, followed by T315I, H396P, native Bcr-Abl and M351T. Similar results were seen in semi-quantitative B-cell transformation assays, where Y253F and E255K showed the most rapid outgrowth, while M351T was slowest. In contrast, when compared to native Bcr-Abl,in myeloid colony formation assays Y253F and E255K showed comparable and M351T and H396P reduced transformation potency, while the potency of T315I was reduced only in the absence of cytokines. The approximate consensus ranking of transformation potency from the different in vitro assays was Y253F > E255K > native Bcr-Abl > T315I > H396P > M351T.These differences were partially reflected in a murine leukemia model, where we observed trends for longer survival in recipients of H396P infected marrow and shorter survival in recipients of E255K. Next, we assayed kinetic properties of the purified KDs and recombinant full length Bcr-Abl in in vitro kinase assays. For the isolated KD, the Kcat/Km of the Y253F mutant was increased over native Bcr-Abl, E255K was similar and the other mutants were slightly (H396P) or significantly (M351T and T315I) reduced. Results with full-length proteins were similar for Y253F, T315I and M351T, while Kcat/Km for E255K was slightly reduced and for H396P slightly increased. Thus, the transformation potency of Y253F, M351T and H396P correlated with intrinsic kinase activity, while the biological activity of E255K and T315I was greater than would be predicted based on kinase assay results. We hypothesized that differential activation of signaling pathways may further modulate transformation potency. Indeed, phosphotyrosine proteome analysis by mass spectrometry revealed differential phosphorylation among the mutants, consistent with altered substrate specificity and pathway activation. For example, phosphorylation of Y1021 of SHIP1 was detected in Ba/F3 cells expressing the M351T and H396P mutant but not in cells expressing other KD mutants or native Bcr-Abl. This was confirmed by immunoblot analysis. Additional experiments showed significant differences by genome-wide microarray expression profiling, consistent with differential activation of signaling pathways. Conclusion: Mutations in the KD of Bcr-Abl influence kinase activity and signaling in a complex fashion, the net results of which is gain or loss of function variants. Drug resistance and transformation potency of mutants may determine the outcome of patients on therapy with Abl kinase inhibitors.

scholarly journals Site-directed mutagenesis of rat muscle 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase: role of Asp-130 in the 2-kinase domain

1994 ◽  
Vol 300 (1) ◽  
pp. 111-115 ◽  
Author(s):  
M H Rider ◽  
K M Crepin ◽  
M De Cloedt ◽  
L Bertrand ◽  
L Hue
Keyword(s):  
Escherichia Coli ◽  
Skeletal Muscle ◽  
Fold Increase ◽  
Kinase Domain ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Kinetic Properties ◽  
Wild Type ◽  
Fluorescence Measurements

Asp-130 of the recombinant skeletal-muscle 6-phosphofructo-2-kinase (PFK-2)/fructose-2,6-bisphosphatase was mutated into Ala in order to study its role in catalysis and/or substrate binding. The D130A mutant displayed a 30- to 140-fold decreased 2-kinase Vmax, depending on the pH, and a 30- and 60-fold increase in Km for MgATP and Fru-6-P respectively at pH 8.5 compared with the wild-type. Mutagenesis of Asp-130 to Ala had no effect on the 2-phosphatase activity, and fluorescence measurements indicated that the changes in kinetic properties of PFK-2 in the D130A mutant were not due to instability. The role of Asp-130 in the 2-kinase reaction is discussed and compared with that of Asp-103 of 6-phosphofructo-1-kinase from Escherichia coli, which binds Mg2+.

Chromatographic Isolation of the LDH-Isoenzymes of Human Blood Platelets and an Investigation of Their Enzyme Kinetics

1968 ◽  
Vol 20 (03/04) ◽  
pp. 301-313 ◽  
Author(s):  
W Schneider ◽  
K Schumacher ◽  
B Thiede ◽  
R Gross
Keyword(s):  
Human Blood ◽  
Blood Platelets ◽  
Deae Cellulose ◽  
Order Reaction ◽  
Kinetic Properties ◽  
Ldh Isoenzymes ◽  
Kinetic Investigations ◽  
Substrate Concentrations ◽  
Chromatographic Isolation ◽  
Human Blood Platelets

SummaryThe LDH-isoenzymes of human blood platelets show a distinct predominance of the isoenzymes 2 and 3 upon chromatography on DEAE-cellulose. Small amounts of LDH-1 are also present, while only traces of LDH-4 and -5 can be detected.Enzyme kinetic investigations of the principal isoenzymes LDH-1, -2 and -3 clearly show that the differences in inhibition constants with pyruvate as substrate which are demonstrable at 25° largely disappear at 37°. On the other hand, the differences among the isoenzymes in their affinity for pyruvate and lactate as substrate as well as in with respect to the optimal substrate concentrations of pyruvate are more marked at 37° than at 25°. Also, the type of inhibition found with lactate as substrate is increasingly the expression of a higher order reaction in going from LDH-1 to LDH-3. A dependence of the LDH distribution pattern upon the metabolism of the cell is discussed. A comparison of our results with thrombocytes with those of other workers with erythrocytes and leucocytes makes it unlikely that the LDH pattern is directly dependent upon the existence of an oxidative metabolism. Rather, the redox potential of the cell could be of importance for the nature of the pattern of isoenzymes and for their differing kinetic properties.

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