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Roles of static and dynamic domains in stability and catalysis of adenylate kinase.

ABSTRACT: Protein dynamics, including conformational switching, are recognized to be crucial for the function of many systems. These motions are more challenging to study than simple static structures. Here, we present evidence suggesting that in the enzyme adenylate kinase large "hinge bending" motions closely related to catalysis are regulated by intrinsic properties of the moving domains and not by their hinges, by anchoring domains, or by remote allosteric-like regions. From a pair of highly homologous mesophilic and thermophilic adenylate kinases, we generated a series of chimeric enzymes using a previously undescribed method with synthetic genes. Subsequent analysis of the chimeras has revealed unexpected spatial separation of stability and activity control. Our results highlight specific contributions of dynamics to catalysis in adenylate kinase. Furthermore, the overall strategy and the specific mutagenesis method used in this study can be generally applied.

SUBMITTER: Bae E

PROVIDER: S-EPMC1413696 | biostudies-literature | 2006 Feb

REPOSITORIES: biostudies-literature

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Roles of static and dynamic domains in stability and catalysis of adenylate kinase.

Bae Euiyoung E Phillips George N GN

Proceedings of the National Academy of Sciences of the United States of America 20060201 7

Protein dynamics, including conformational switching, are recognized to be crucial for the function of many systems. These motions are more challenging to study than simple static structures. Here, we present evidence suggesting that in the enzyme adenylate kinase large "hinge bending" motions closely related to catalysis are regulated by intrinsic properties of the moving domains and not by their hinges, by anchoring domains, or by remote allosteric-like regions. From a pair of highly homologou ...[more]

PMID: 16452168

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Project description:BackgroundIt has been demonstrated that the adenyl moiety of ATP plays a direct role in the regulation of ATP binding and/or phosphoryl transfer within a range of kinase and synthetase enzymes. The role of the C8-H of ATP in the binding and/or phosphoryl transfer on the enzyme activity of a number of kinase and synthetase enzymes has been elucidated. The intrinsic catalysis rate mediated by each kinase enzyme is complex, yielding apparent KM values ranging from less than 0.4 μM to more than 1 mM for ATP in the various kinases. Using a combination of ATP deuterated at the C8 position (C8D-ATP) as a molecular probe with site directed mutagenesis (SDM) of conserved amino acid residues in shikimate kinase and adenylate kinase active sites, we have elucidated a mechanism by which the ATP C8-H is induced to be labile in the broader kinase family. We have demonstrated the direct role of the C8-H in the rate of ATP consumption, and the direct role played by conserved Thr residues interacting with the C8-H. The mechanism by which the vast range in KM might be achieved is also suggested by these findings.ResultsWe have demonstrated the mechanism by which the enzyme activities of Group 2 kinases, shikimate kinase (SK) and adenylate kinase 1 (AK1), are controlled by the C8-H of ATP. Mutations of the conserved threonine residues associated with the labile C8-H cause the enzymes to lose their saturation kinetics over the concentration range tested. The relationship between the role C8-H of ATP in the reaction mechanism and the ATP concentration as they influence the saturation kinetics of the enzyme activity is also shown. The SDM clearly identified the amino acid residues involved in both the catalysis and regulation of phosphoryl transfer in SK and AK1 as mediated by C8H-ATP.ConclusionsThe data outlined serves to demonstrate the "push" mechanism associated with the control of the saturation kinetics of Group 2 kinases mediated by ATP C8-H. It is therefore conceivable that kinase enzymes achieve the observed 2,500-fold variation in KM through a combination of the various conserved "push" and "pull" mechanisms associated with the release of C8-H, the proton transfer cascades unique to the class of kinase in question and the resultant/concomitant creation of a pentavalent species from the γ-phosphate group of ATP. Also demonstrated is the interplay between the role of the C8-H of ATP and the ATP concentration in the observed enzyme activity. The lability of the C8-H mediated by active site residues co-ordinated to the purine ring of ATP therefore plays a significant role in explaining the broad KM range associated with kinase steady state enzyme activities.

Project description:IntroductionGlobally, 300 million adults have clinical obesity. Heightened adiposity and inadequate musculature secondary to obesity alter bipedal stance and gait, diminish musculoskeletal tissue quality, and compromise neuromuscular feedback; these physiological changes alter stability and increase injury risk from falls. Studies in the field focus on obese patients across a broad range of body mass indices (BMI >30 kg/m2) but without isolating the most morbidly obese subset (BMI ≥40 kg/m2). We investigated the impact of obesity in perturbing postural stability in morbidly obese subjects elected for bariatric intervention, harboring a higher-spectrum BMI.Subjects and methodsTraditional force plate measurements and stabilograms are gold standards employed when measuring center of pressure (COP) and postural sway. To quantify the extent of postural instability in subjects with obesity before bariatric surgery, we assessed 17 obese subjects with an average BMI of 40 kg/m2 in contrast to 13 nonobese subjects with an average BMI of 30 kg/m2. COP and postural sway were measured from static and dynamic tasks. Involuntary movements were measured when patients performed static stances, with eyes either opened or closed. Two additional voluntary movements were measured when subjects performed dynamic, upper torso tasks with eyes opened.ResultsMean body weight was 85% (p < 0.001) greater in obese than nonobese subjects. Following static balance assessments, we observed greater sway displacement in the anteroposterior (AP) direction in obese subjects with eyes open (87%, p < 0.002) and eyes closed (76%, p = 0.04) versus nonobese subjects. Obese subjects also exhibited a higher COP velocity in static tests when subjects' eyes were open (47%, p = 0.04). Dynamic tests demonstrated no differences between groups in sway displacement in either direction; however, COP velocity in the mediolateral (ML) direction was reduced (31%, p < 0.02) in obese subjects while voluntarily swaying in the AP direction, but increased in the same cohort when swaying in the ML direction (40%, p < 0.04).Discussion and conclusionImportantly, these data highlight obesity's contribution towards increased postural instability. Obese subjects exhibited greater COP displacement at higher AP velocities versus nonobese subjects, suggesting that clinically obese individuals show greater instability than nonobese subjects. Identifying factors contributory to instability could encourage patient-specific physical therapies and presurgical measures to mitigate instability and monitor postsurgical balance improvements.

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Roles of static and dynamic domains in stability and catalysis of adenylate kinase.

Publications

Roles of static and dynamic domains in stability and catalysis of adenylate kinase.

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