Mechanism of adenylate kinase. The conserved aspartates 140 and 141 are important for transition state stabilization instead of substrate-induced conformational changes.

The goal of this work is to evaluate the quantitative contribution of Asp-140 and Asp-141 to conformational changes and/or substrate stabilization in the catalysis by chicken muscle adenylate kinase (AK), by use of kinetic and structural analysis of the single alanine mutants D140A and D141A as well as the salt bridge double mutants R138M,D140A and R132M,D141A. The single mutants D140A and D141A displayed small increases in Km (6-12-fold) and moderate decreases in kcat (17-78-fold). Kinetic analysis with the slowly reacting substrate system MgATP and dAMP suggested that the decrease in kcat is not caused by a decrease in the conformational step(s) relative to the chemical step. Most of the kinetic constants of the double mutants R138M,D140A and R132M,D141A are similar to those of the single mutants R138M and R132M, respectively. Detailed analysis by two-dimensional NMR indicated no appreciable changes in the conformations of the free enzyme or the complex with MgAP5A (where AP5A is P1,P5-bis(5'-adenosyl)pentaphosphate), for both single and double mutants. These results taken together suggest that Asp-140 and Asp-141 are unimportant for substrate-induced conformational changes in AK; their roles are mainly to assist Arg-138 and Arg-132 in stabilizing the transition state. The structural results also suggest that AK is a flexible enzyme.

Pseudomonas aeruginosa AlgB, which modulates the expression of alginate, is a member of the NtrC subclass of prokaryotic regulators.

The Pseudomonas aeruginosa exopolysaccharide alginate is an important virulence factor in chronic pulmonary infections of cystic fibrosis patients. We determined the nucleotide sequence of the gene, algB, which regulates the level of exopolysaccharide produced by mucoid P. aeruginosa. The predicted amino acid sequence of AlgB revealed a high degree of similarity to the regulatory proteins in the NtrC subclass of 'two-component regulatory systems'. AlgB expression in Escherichia coli minicells showed a molecular weight of approximately 50,000 Da, comparable to that of the inferred amino acid sequence (49,318 Da). We show that algB is transcriptionally active in mucoid strains of P. aeruginosa and regulates the expression of the alginate biosynthetic gene, algD, thereby resulting in increased expression of alginate in mucoid P. aeruginosa.

Mechanism of adenylate kinase. Critical evaluation of the X-ray model and assignment of the AMP site.

The substrate binding sites of adenylate kinase (AK) proposed by X-ray crystallographic studies [Pai, E. F., Sachsenheimer, W., Schirmer, R. H., & Schulz, G. E. (1977) J. Mol. Biol. 114, 37-45, and subsequent revisions] were evaluated by site-specific mutagenesis in conjunction with structural analysis by NMR. The residues examined in this report include two near an adenosine site (threonine-39 and arginine-44) and two in the phosphate binding region (arginine-128 and arginine-149). The results and conclusions are summarized as follows: (a) Although Thr-39 is very close to an adenine site [Egner, U., Tomasselli, A. G., & Schulz, G. E. (1987) J. Mol. Biol. 195, 649-658], it is nonessential either structurally or functionally. (b) The R44M mutant enzyme showed significant increases in the Michaelis and dissociation constants of adenosine 5'-monophosphate (AMP) (36- and 22-fold, respectively) while all other kinetic parameters were relatively unperturbed. The proton NMR property of this mutant was unchanged in the free enzyme and only slightly perturbed in the binary complexes with AMP and with MgATP (adenosine 5'-triphosphate), and in the ternary complex with MgAP5A [P1,P5-bis(5'-adenosyl) pentaphosphate]. These results indicate that Arg-44 interacts specifically with AMP starting at the binary complex, and suggest that the MgATP site proposed by Pai et al. (1977) is likely to be the AMP site. (c) The kinetic parameters of R149M were dramatically perturbed: kcat decreased by a factor of 1540, Km increased to 130-fold, and kcat/Km decreased by a factor of 2 X 10(5).(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanism of adenylate kinase. Structural and functional roles of the conserved arginine-97 and arginine-132.

The structural and functional roles of two conserved active site residues, Arg-97 and Arg-132, in chicken muscle adenylate kinase (AK) were evaluated by site-directed mutagenesis in conjunction with one- and two-dimensional proton nuclear magnetic resonance (NMR), kinetics, and guanidine hydrochloride-induced denaturation. In addition, 31P NMR analysis was used to evaluate the contribution of Arg-97 to the phosphorus stereospecificity of AK. The results and conclusions are summarized as follows: (i) Kinetic analysis of R97M reveals 6- and 28-fold increases in the dissociation constant Ki and Michaelis constant K of AMP, respectively, and a moderate 30-fold decrease in kcat. The Ki and K values of MgATP are relatively unperturbed. The localized effect of AMP stabilization was independently confirmed by proton NMR titration, which showed a ca. 20-fold increase in the dissociation constant of AMP but not of MgATP. (ii) R132M affords a dramatic decrease in kcat by a factor of 8.0 x 10(3), with unchanged dissociation and Michaelis constants for either substrate. The lack of perturbation in the affinities toward substrates was confirmed by proton NMR titration. (iii) Although small chemical shift changes were observed for the free mutants and their complexes with substrates, further analyses by nuclear Overhauser enhanced spectroscopy with the bisubstrate analogue inhibitor, P1,P5-bis(5'-adenosyl)pentaphosphate (AP5A), indicated little perturbation in the global conformation. (iv) Contributions to conformational stability by Arg-97 and Arg-132 are negligible on the basis of the free energy of unfolding, delta GdH2O. (v) R97M was predicted and demonstrated to exhibit enhanced stereospecificity at the AMP site by at least 10-fold relative to WT in the conversion of adenosine 5'-monothiophosphate to adenosine 5'-(1-thiodiphosphate). This result for R97M was predicted on the basis of the orientation of Arg-97 relative to Arg-44 and AMP in the active site as observed in available crystal structures and the stereospecificity results of R44M [Jiang, R.-T., Dahnke, T., & Tsai, M.-D. (1991) J. Am. Chem. Soc. 113, 5485-5486]. (vi) The above structural and functional analyses led us to conclude that Arg-97 interacts with the phosphoryl group of AMP, beginning at the binary complex (1-2 kcal/mol), continuing through the transition state (3.5 kcal/mol), and that Arg-132 stabilizes the transition state by greater than 5 kcal/mol. (vii) The functional importance of Arg-97 appears to be similar to that of Arg-44 [Yan, H., Dahnke, T., Zhou, B., Nakazawa, A., & Tsai, M.-D. (1990) Biochemistry 29, 10956-10964].(ABSTRACT TRUNCATED AT 400 WORDS)