Mechanism of adenylate kinase. Histidine-36 is not directly involved in catalysis, but protects cysteine-25 and stabilizes the tertiary structure

Tian, Gaochao; Sanders, Charles R.; Kishi, Fumio; Nakazawa, Atsushi; Tsai, Ming‐Daw

doi:10.1021/bi00415a024

Cited by 34 publications

(47 citation statements)

References 51 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Our site-directed mutagenesis studies with chicken muscle AK (cAK)* have shown that, contrary to suggestions from previous NMR studies (Fry et al, 1985;Mildvan & Fry, 1987), Lys-27 and His-36 are not important catalytically (Tian et al, 1988(Tian et al, , 1990. The catalytic role of the absolutely conserved Lys-21 was suggested to be uncertain despite a large decrease in the k,, of K21M due to significant changes in the proton NMR spectrum of this mutant (Tian et al, 1990).…”

contrasting

confidence: 84%

Mechanism of adenylate kinase. Structural and functional demonstration of arginine-138 as a key catalytic residue that cannot be replaced by lysine

1990

Self Cite

View full text Add to dashboard Cite

Replacement of the arginine-138 of adenylate kinase (AK) by lysine or methionine resulted in a decrease in kcat by a factor of 10(4), increases in Km by a factor of 10-20, and relatively little changes in dissociation constants. Proton nuclear magnetic resonance (NMR) studies were then undertaken to obtain structural information for quantitative interpretation of the kinetic data. Since the lysine mutant (R138K) represents a conservative mutation with surprisingly large effects on kinetics, structural studies were focused on the wild type (WT) and R138K. The results and conclusions are summarized as follows: (i) The aromatic spin systems of WT and R138K were assigned from total correlated spectroscopy (TOCSY). Comparison of the chemical shifts of aromatic protons, one-dimensional spectra, TOCSY, and nuclear Overhauser enhanced spectroscopy (NOESY) indicated that the conformation of R138K was almost unperturbed relative to that of WT. Thus Arg-138 is not important for the tertiary structure. (ii) Proton NMR titrations with AMP and MgATP suggested that substrate binding affinities and substrate-induced conformational changes are nearly identical between WT and R138K. Thus arginine-138 should not be involved in stabilizing the first substrate in the binary complex. (iii) Notable differences were observed between the proton NMR spectra of the WT and R138K complexes with the reaction mixture, which agrees with the perturbation in the Km values of R138K. The differences were analyzed in detail by using a "static reaction mixture'--p1, p5-bis(5'-adenosyl)pentaphosphate (MgAP5A). The aromatic spin systems of WT + MgAP5A and R138K + MgAP5A were partially assigned from various two-dimensional spectra.(ABSTRACT TRUNCATED AT 250 WORDS)

show abstract

contrasting

confidence: 84%

Mechanism of adenylate kinase. Structural and functional demonstration of arginine-138 as a key catalytic residue that cannot be replaced by lysine

1990

Self Cite

View full text Add to dashboard Cite

show abstract

“…It is easy to incorporate residue specific energies for these thresholds as in the work of Munoz and Eason (10). The unfolding free energy of the adenylate kinase from Escherichia coli has been reported to be Ϸ4 kcal͞mol for guanidine hydrochloride denaturation (30) and Ϸ10 kcal͞mol for urea denaturation (31). Heat denaturation of adenylate kinase from Saccharomyces cerevisiae is reported to give a stability of Ϸ4 kcal͞mol (32).…”

Section: Nonlinear Elastic Modelsmentioning

confidence: 99%

Nonlinear elasticity, proteinquakes, and the energy landscapes of functional transitions in proteins

Miyashita

Onuchic

Wolynes

2003

Proc. Natl. Acad. Sci. U.S.A.

471

575

View full text Add to dashboard Cite

Large-scale motions of biomolecules involve linear elastic deformations along low-frequency normal modes, but for function nonlinearity is essential. In addition, unlike macroscopic machines, biological machines can locally break and then reassemble during function. We present a model for global structural transformations, such as allostery, that involve large-scale motion and possible partial unfolding, illustrating the method with the conformational transition of adenylate kinase. Structural deformation between open and closed states occurs via low-frequency modes on separate reactant and product surfaces, switching from one state to the other when energetically favorable. The switching model is the most straightforward anharmonic interpolation, which allows the barrier for a process to be estimated from a linear normal mode calculation, which by itself cannot be used for activated events. Local unfolding, or cracking, occurs in regions where the elastic stress becomes too high during the transition. Cracking leads to a counterintuitive catalytic effect of added denaturant on allosteric enzyme function. It also leads to unusual relationships between equilibrium constant and rate like those seen recently in singlemolecule experiments of motor proteins.T he regulation of biological machinery through allostery is a dominant theme in our modern molecular understanding of life. Allostery requires a biomolecule to have at least a pair or, more likely, a multiplicity of conformational states of nearly equal free energy. How can we describe movement between such states? When the pair of states exhibits large-scale structural differences, it is tempting to connect the states by routes using the low-frequency collective elastic vibrations around each structure, the normal modes with the smallest restoring forces. Even in its simplest form, the notion of normal modes is remarkably successful for visualizing and predicting the character of the motions. The motions are in reality overdamped, but their structure often parallels the low-frequency normal modes (1). Yet clearly, a linear normal mode description cannot be complete because the very existence of the two low-lying conformations requires us to acknowledge considerable anharmonicity. The normal mode picture strictly describes the excitations about a single minimum. The limited adequacy of a normal mode description becomes even more apparent when we try to embed our picture of the motion between two dominant conformational states in the complete energy landscape of a biomolecule, which is replete with a myriad of local minima, ranging from the more subtle conformational substrates apparent in kinetic experiments (2) to the still more disordered states that are partially unfolded. Our goal in this article is to describe how allosteric conformational switches function by using a theoretical framework that unites an energy landscape description with the elastic model based on normal modes. To do so we need to go beyond the usual approaches that describe only the geometr...

show abstract

“…The values of k,,,, K (Michaelis constant), and Ki (dissociation constant) were obtained by varying both MgATP and AMP concentrations followed by analyses according to the equation of Cleland (1986) for a random Bi-Bi system. The details have been described previously (Tian et al, 1988).…”

mentioning

confidence: 99%

Mechanism of adenylate kinase. The essential lysine helps to orient the phosphates and the active site residues to proper conformations

Byeon¹,

Shi²,

Tsai³

1995

Biochemistry

View full text Add to dashboard Cite

Although how Lys21 interacts with the substrate MgATP of muscle adenylate kinase (AK) can now be deduced from the crystal structure of Escherichia coli AK.MgAP5A [P1,P5-bis(5'-adenosyl) pentaphosphate] [Müller, C. W., & Schulz, G. E. (1992) J. Mol. Biol. 224, 159-177], its contribution to catalysis has not yet been demonstrated by functional studies since the proton NMR of the K21M mutant was shown to be perturbed significantly [Tian, G., Yan., H., Jiang, R.-T., Kishi, F., Nakazawa, A., & Tsai, M.-D. (1990) Biochemistry 29, 4296-4304]. We therefore undertook further structural and functional analyses of a conservative mutant K21R and a nonconservative mutant K21A. In addition to kinetic analyses, the structures of the mutants were analyzed by one- and two-dimensional proton NMR spectroscopy and (1H, 15N) heteronuclear multiple-quantum coherence (HMQC) experiments. Detailed assignments were performed in reference to the total backbone assignments of the WT AK.MgAP5A complex [Byeon, I.-J. L., Yan, H., Edison, A. S., Mooberry, E. S., Abildgaard, F., Markley, J. L., & Tsai, M.-D. (1993) Biochemistry 32, 12508-12521]. The analysis showed that the residues located near the active site (Gly15, Thr23, Arg97, Gln101, Arg128, Arg132, Asp140, Asp141, and Tyr153) exhibit greater changes in 1H-15N chemical shifts. Finally, two-dimensional 31P-31P COSY experiments were used to examine the effects of the lysine side chain on the phosphate groups in the bound AP5A. Our data have led to the following conclusions independent of the crystal structure: (i) Because the perturbations in the conformation of the mutants are not global and are mainly localized at active site residues and Tyr153, the side chain of Lys21 can be concluded to stabilize the transition state in the catalysis of AK by up to 7 kcal/mol on the basis of the 10(5)-fold decreases in the kcat/Km of mutants. (ii) The results of 31P NMR analyses suggest that Lys21 functions by orienting the triphosphate chain of MgATP to a proper conformation required for catalysis. (iii) The interaction between Lys21 and the phosphate chain in turn dictates the interactions between the substrates and the active site residues. In the K21R.MgATP complex, the NH chemical shifts of many of the active site residues are perturbed. (iv) The catalytic functions of Lys21 cannot be replaced by a conservative residue arginine. In addition, since K21A and K21R behave similarly, the catalytic function of Lys21 should not be merely a charge effect.

show abstract

Mechanism of adenylate kinase. Histidine-36 is not directly involved in catalysis, but protects cysteine-25 and stabilizes the tertiary structure

Cited by 34 publications

References 51 publications

Mechanism of adenylate kinase. Structural and functional demonstration of arginine-138 as a key catalytic residue that cannot be replaced by lysine

Mechanism of adenylate kinase. Structural and functional demonstration of arginine-138 as a key catalytic residue that cannot be replaced by lysine

Nonlinear elasticity, proteinquakes, and the energy landscapes of functional transitions in proteins

Mechanism of adenylate kinase. The essential lysine helps to orient the phosphates and the active site residues to proper conformations

Contact Info

Product

Resources

About