Hydrogen bonding and biological specificity analysed by protein engineering

Fersht, Alan R.; Shi, Jianping; Knill-Jones, J.W.; Lowe, Denise M.; Wilkinson, Anthony J.; Blow, D. M.; Brick, P.; Carter, Paul; Waye, Mary Miu Yee; Winter, Greg

doi:10.1038/314235a0

Cited by 1,103 publications

(769 citation statements)

References 19 publications

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“…These salt bridges appear important to maintain this FA binding site because hydrogen bonds between oppositecharged residues can be much stronger (4.5 Kcal/ mol) than regular hydrogen bonds (2-3 Kcal/mol). 25 These salt bridges network could also be a mechanism to regulate the ligand loading and unloading. There is one hydrogen bond between DAUDA 6 and rHSA between the oxygen atom of rHSA Asp324 to the dansyl group (N1) of DUADA.…”

Section: Reliability Of the Rhsa-myr-dauda Structurementioning

confidence: 99%

A fluorescent fatty acid probe, DAUDA, selectively displaces two myristates bound in human serum albumin

et al. 2011

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11-(Dansylamino) undecanoic acid (DAUDA) is a dansyl-type fluorophore and has widely used as a probe to determine the binding site for human serum albumin (HSA). Here, we reported that structure of HSA-Myristate-DAUDA ternary complex and identified clearly the presence of two DAUDA molecules at fatty acid (FA) binding site 6 and 7 of HSA, thus showing these two sites are weak FA binding sites. This result also show that DAUDA is an appropriate probe for FA site 6 and 7 on HSA as previous studied, but not a good probe of FA binding site 1 that is likely bilirubin binding site on HSA.

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Section: Reliability Of the Rhsa-myr-dauda Structurementioning

confidence: 99%

A fluorescent fatty acid probe, DAUDA, selectively displaces two myristates bound in human serum albumin

et al. 2011

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“…For the fluorescence decrease observed in the amino acid activation reaction, this equation is: (11) where F is the relative fluorescence at time t, F o is the initial relative fluorescence value, k obs is the observed rate constant, t is the time, and C is the offset of the curve from zero. For the fluorescence increase observed in either the reverse amino acid activation reaction (E•Tyr~AMP + PP i ⇌ E + Tyr + ATP) or the transfer of the tyrosyl moiety to tRNA (E•Tyr~AMP + tRNA ⇌ E + Tyr~tRNA + AMP), the following equation is used: (12) where F ∞ is the final relative fluorescence value, and F, k obs , , t, and C are as defined above.…”

Section: A General Considerationsmentioning

confidence: 99%

“…While less broadly applied, pre-steady state experiments have provided valuable insights into catalytic mechanisms in many aaRS systems (summarized in Table 1), often allowing the determination of the thermodynamic and kinetic contributions of particular enzyme-substrate interactions to specific steps and energetic barriers along a reaction path [11]. The most common pre-steady state kinetic approaches for the aaRSs are rapid chemical quench assays, which follow rates of product formation, and stopped-flow fluorimetry, which takes advantage of changes in the intrinsic tryptophan fluorescence that are correlated with the reaction chemistry.…”

Section: Introductionmentioning

confidence: 99%

Methods for kinetic and thermodynamic analysis of aminoacyl-tRNA synthetases

Francklyn

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Perona

et al. 2008

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The accuracy of protein synthesis relies on the ability of aminoacyl-tRNA synthetases (aaRSs) to discriminate among true and near cognate substrates. To date, analysis of aaRSs function, including identification of residues of aaRS participating in amino acid and tRNA discrimination, has largely relied on the steady state kinetic pyrophosphate exchange and aminoacylation assays. Pre-steady state kinetic studies investigating a more limited set of aaRS systems have also been undertaken to assess the energetic contributions of individual enzyme-substrate interactions, particularly in the adenylation half reaction. More recently, a renewed interest in the use of rapid kinetics approaches for aaRSs has led to their application to several new aaRS systems, resulting in the identification of mechanistic differences that distinguish the two structurally distinct aaRS classes. Here, we review the techniques for thermodynamic and kinetic analysis of aaRS function. Following a brief survey of methods for the preparation of materials and for steady state kinetic analysis, this review will describe pre-steady state kinetic methods employing rapid quench and stopped-flow fluorescence for analysis of the activation and aminoacyl transfer reactions. Application of these methods to any aaRS system allows the investigator to derive detailed kinetic mechanisms for the activation and aminoacyl transfer reactions, permitting issues of substrate specificity, stereochemical mechanism, and inhibitor interaction to be addressed in a rigorous and quantitative fashion.

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“…The residues of crystal contact-Ia (only in subunit I; see Mittl & Schulz, 1994) are marked by daggers and those in crystal contact-V (both subunits) by circles. Figure 5 shows that the exchanges Arg 198 + Met, Lys 199 -+ Phe, and Arg 204 + Pro render the environment of Glu 197 rather nonpolar, increasing its hydrogen bonding strength appreciably (Fersht et al, 1985). The carboxylate of Glu 197 is poised to bind the adenosine ribose tightly.…”

Section: Phc199mentioning

confidence: 99%

Anatomy of an engineered NAD‐binding site

et al. 1994

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The coenzyme specificity of Escherichia coli glutathione reductase was switched from NADP to NAD by modifying the environment of the 2'-phosphate binding site through a set of point mutations: A179G, A183G, V197E, R198M, K199F, H200D, and R204P (Scrutton NS, Berry A, Perham RN, 1990, Nature 343:38-43). In order to analyze the structural changes involved, we have determined 4 high-resolution crystal structures, i.e., the structures of the wild-type enzyme (1.86 A resolution, R-factor of l6.8%), of the wild-type enzyme ligated with NADP (2.OA, 20.8%), of the NAD-dependent mutant (1.74A, 16.8%), and of the NAD-dependent mutant ligated with NAD (2.2 A, 16.9%). A comparison of these structures reveals subtle differences that explain details of the specificity change. In particular, a peptide rotation occurs close to the adenosine ribose, with a concomitant change of the ribose pucker. The mutations cause a contraction of the local chain fold. Furthermore, the engineered NADbinding site assumes a less rigid structure than the NADP site of the wild-type enzyme. A superposition of the ligated structures shows a displacement of NAD versus NADP such that the electron pathway from the nicotinamide ring to FAD is elongated, which may explain the lower catalytic efficiency of the mutant. Because the nicotinamide is as much as 15 A from the sites of the mutations, this observation reminds us that mutations may have important long-range consequences that are difficult to anticipate.Keywords: coenzyme specificity change; glutathione reductase; NAD-binding site; NADP-binding site; protein engineeringThe coenzyme NADP differs from NAD by virtue of its additional 2"phosphate at the adenosine ribose. These ubiquitous dinucleotides undergo redox reactions with distinct metabolic functions. NAD participates mostly in catabolic reactions, for instance, NADH is a fuel for the regeneration of ATP in oxidative phosphorylation. In contrast, NADPH provides redox equivalents for anabolic reactions, as for example in lipid biosynthesis. In keeping with this, glutathione reductase from Escherichia coli (EC 1.6.4.2) is an FAD-dependent homodimeric enzyme with an M, of 49,560 per subunit that catalyzes the reduction of oxidized glutathione at the expense of NADPH according to the equation: NADPH + GSSG + H+ e NADP' + 2GSH. Scrutton et al. (1990) created an NAD-dependent mutant of GR,,, by the introduction of 7 point mutations (A179G, A183G, V197E, R198M, K199F, H200D, and R204P) in the Reprint requests to:

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Hydrogen bonding and biological specificity analysed by protein engineering

Cited by 1,103 publications

References 19 publications

A fluorescent fatty acid probe, DAUDA, selectively displaces two myristates bound in human serum albumin

A fluorescent fatty acid probe, DAUDA, selectively displaces two myristates bound in human serum albumin

Methods for kinetic and thermodynamic analysis of aminoacyl-tRNA synthetases

Anatomy of an engineered NAD‐binding site

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