Identification of residues involved in a conformational change accompanying substitutions for glutamate-43 in staphylococcal nuclease

Wilde, Joyce A.; Bolton, Philip H.; Dell’Acqua, Mark L.; Hibler, David W.; Pourmotabbed, Tayebeh; Gerlt, J.A.

doi:10.1021/bi00411a033

Cited by 42 publications

(37 citation statements)

References 13 publications

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“…However, in this respect, it is important to clarify a major misconception that emerged from some such studies (e.g. Hale et al 1993; Wilde et al 1998), that mutated the general base, Glu 43 , to an aspartate. Although these works found a reduction in the catalytic effect of the enzyme by a factor of 2000, which (as outlined in chapter 9 of Warshel, 1991) is fully consistent with the increase in distance to the base, it has been argued that the observed effect does not prove that Glu 43 is the base, since the mutation involves significant structural changes.…”

Section: Enzymatic Phosphoryl Transfermentioning

confidence: 99%

Why nature really chose phosphate

Kamerlin

Sharma

Prasad

et al. 2013

Quart. Rev. Biophys.

319

448

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Phosphoryl transfer plays key roles in signaling, energy transduction, protein synthesis, and maintaining the integrity of the genetic material. On the surface, it would appear to be a simple nucleophile displacement reaction. However, this simplicity is deceptive, as, even in aqueous solution, the low-lying d-orbitals on the phosphorus atom allow for eight distinct mechanistic possibilities, before even introducing the complexities of the enzyme catalyzed reactions. To further complicate matters, while powerful, traditional experimental techniques such as the use of linear free-energy relationships (LFER) or measuring isotope effects cannot make unique distinctions between different potential mechanisms. A quarter of a century has passed since Westheimer wrote his seminal review, ‘Why Nature Chose Phosphate’ (Science 235 (1987), 1173), and a lot has changed in the field since then. The present review revisits this biologically crucial issue, exploring both relevant enzymatic systems as well as the corresponding chemistry in aqueous solution, and demonstrating that the only way key questions in this field are likely to be resolved is through careful theoretical studies (which of course should be able to reproduce all relevant experimental data). Finally, we demonstrate that the reason that naturereallychose phosphate is due to interplay between two counteracting effects: on the one hand, phosphates are negatively charged and the resulting charge-charge repulsion with the attacking nucleophile contributes to the very high barrier for hydrolysis, making phosphate esters among the most inert compounds known. However, biology is not only about reducing the barrier to unfavorable chemical reactions. That is, the same charge-charge repulsion that makes phosphate ester hydrolysis so unfavorable also makes it possible to regulate, by exploiting the electrostatics. This means that phosphate ester hydrolysis can not only be turned on, but also be turned off, by fine tuning the electrostatic environment and the present review demonstrates numerous examples where this is the case. Without this capacity for regulation, it would be impossible to have for instance a signaling or metabolic cascade, where the action of each participant is determined by the fine-tuned activity of the previous piece in the production line. This makes phosphate esters the ideal compounds to facilitate life as we know it.

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Section: Enzymatic Phosphoryl Transfermentioning

confidence: 99%

Why nature really chose phosphate

Kamerlin

Sharma

Prasad

et al. 2013

Quart. Rev. Biophys.

319

448

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“…However, the potential interactions between amino acid residues, with the exception of charge-charge interactions, are strongly distance-dependent (41). Also, the effects of amino acid substitutions on protein structure are often localized to the immediate vicinity of the substitution (10,(42)(43)(44)(45) used to study the effects of substitutions on other properties of the gene V protein such as resistance to irreversible thermal denaturation (31) or folding and unfolding rates (30) in the context of WT stability and DNA binding affinity. Protein engineering through the combination of singlesubstitution mutants may be most successful at adjusting those properties of concern in vitro, rather than in vivo activity.…”

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confidence: 99%

Engineering multiple properties of a protein by combinatorial mutagenesis.

Sandberg

Terwilliger

1993

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

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A method for simultaneously engineering multiple properties of a protein, based on the observed additivity of effects of individual mutations, b presented. We show that, for the gene V protein of bacteriophage n, effects of double mutations on both protein stability and DNA binding ainity are approximately equal to the sums ofthe effects ofthe consituent single mutations. This additivity of effects implies that it Is posible to deliberately construct mutant proteins optimizd for multiple properties by combination of appropriate single mutations chosen from a characterized library.One of the long-term goals of the study of mutational effects on protein stability and activity is to devise a method by which mutations can be rationally employed to alter or "engineer" the properties of proteins with predictable results. Recombinant DNA technology has allowed the construction of proteins of altered stability in vitro (1-16), catalytic efficiency (17-19), substrate specificity (20-23), and resistance to in vivo thermal inactivation (24) through the use of single or multiple amino acid substitutions. This effort has been greatly helped by the fact that the effects of amino acid substitutions on such properties of proteins tend to be additive as mutations accumulate, provided that the substituting residues do not interact functionally or by direct contact (25). For example, additive increases in the stability of subtilisin BPN' have been achieved by combining mutations at six sites in the protein tertiary structure (16). The six mutations individually stabilize the protein by 0.3-1.3 kcal/ mol, and the individual effects sum to a stability increase of 3.8 kcal/mol predicted for the hexa-mutant. The observed stabilization of the mutant containing all six substitutions is 4.3 kcal/mol (16). Additive effects ofamino acid substitutions have been used to engineer incremental increases in the stability of other proteins including the N-terminal domain of A repressor (5, 13), T4 lysozyme (9, 12), kanamycin nucleotidyltransferase (6), and neutral protease (26) as well. This strategy of additive mutation has also been employed to alter binding affinities or specificities of proteins, such as A repressor (20), subtilisin (21-23), and glutathione reductase (27), for their substrates or cofactors, and to alter the pH profile of subtilisin (17).A factor complicating the effort to engineer proteins by mutation is that most single amino acid substitutions alter multiple properties ofthe proteins in which they are made. To be functional, a protein must be at once stable, yet flexible, with high catalytic activity balanced against substrate specificity. Because mutants affecting only one of these properties are relatively rare, it appears difficult to optimize one characteristic of a protein through mutations while maintaining adequacy in the others. However, the observation that mutational effects on the in vitro properties of proteins areThe publication costs of this article were defrayed in part by page charge payment. This ar...

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“…Tian et al (1990). a particular residue is not involved in catalysis if the kinetic properties of its mutants are not perturbed, it is difficult to unequivocally identify catalytically important residues and to quantitatively evaluate their contributions to the transitionstate stabilization unless it can be demonstrated that the structure of the mutant is not perturbed (Wilde et al, 1988).…”

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confidence: 99%

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

1990

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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)

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Identification of residues involved in a conformational change accompanying substitutions for glutamate-43 in staphylococcal nuclease

Cited by 42 publications

References 13 publications

Why nature really chose phosphate

Why nature really chose phosphate

Engineering multiple properties of a protein by combinatorial mutagenesis.

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

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