Reaction energetics of a mutant triose phosphate isomerase in which the active-site glutamate has been changed to aspartate

Raines, Ronald T.; Sutton, Eliza L.; Straus, Daniel S.; Gilbert, Walter; Knowles, Jeremy R.

doi:10.1021/bi00370a057

Cited by 118 publications

(104 citation statements)

References 36 publications

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“…Such mutations are typically highly deleterious and have been shown to displace the position of the carboxylate by ∼1 Å relative to its wild-type position in several cases (19)(20)(21)(22)(23), a displacement similar to that observed for the Phe54Gly mutation described above.…”

Section: Double-mutant Cycles To Test the Role Of Phe54 And Phe116 Inmentioning

confidence: 53%

“…Prior studies have used mutations that add or remove a sidechain methylene group to test the importance of positioning active-site carboxylate groups (19)(20)(21)(22)(23)(24). Such mutations are typically highly deleterious and have been shown to displace the position of the carboxylate by ∼1 Å relative to its wild-type position in several cases (19)(20)(21)(22)(23), a displacement similar to that observed for the Phe54Gly mutation described above.…”

Section: Double-mutant Cycles To Test the Role Of Phe54 And Phe116 Inmentioning

confidence: 87%

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Use of anion–aromatic interactions to position the general base in the ketosteroid isomerase active site

Schwans¹,

Sunden²,

Lassila³

et al. 2013

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

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Although the cation-pi pair, formed between a side chain or substrate cation and the negative electrostatic potential of a pi system on the face of an aromatic ring, has been widely discussed and has been shown to be important in protein structure and protein-ligand interactions, there has been little discussion of the potential structural and functional importance in proteins of the related anion-aromatic pair (i.e., interaction of a negatively charged group with the positive electrostatic potential on the ring edge of an aromatic group). We posited, based on prior structural information, that anion-aromatic interactions between the anionic Asp general base and Phe54 and Phe116 might be used instead of a hydrogen-bond network to position the general base in the active site of ketosteroid isomerase from Comamonas testosteroni as there are no neighboring hydrogenbonding groups. We have tested the role of the Phe residues using site-directed mutagenesis, double-mutant cycles, and high-resolution X-ray crystallography. These results indicate a catalytic role of these Phe residues. Extensive analysis of the Protein Data Bank provides strong support for a catalytic role of these and other Phe residues in providing anion-aromatic interactions that position anionic general bases within enzyme active sites. Our results further reveal a potential selective advantage of Phe in certain situations, relative to more traditional hydrogen-bonding groups, because it can simultaneously aid in the binding of hydrophobic substrates and positioning of a neighboring general base.enzyme catalysis | general-base catalysis | noncovalent interactions E nzymes use the same functional groups to achieve "chemical catalysis" as small-molecule catalysts, and yet enzymes attain much greater rate enhancements. A distinguishing feature of enzymes is that their reactions occur in highly specialized active sites that use noncovalent interactions to precisely position enzymatic functional groups relative to substrates and relative to other active-site features. Understanding how these groups are positioned within enzyme active sites is key for understanding the differences between enzymes and small-molecule catalysts.It is generally recognized that hydrophobic interactions can help bind and position substrates in enzyme active sites (1, 2). Beyond hydrophobic interactions, much attention has focused on the importance of hydrogen bonds in precisely positioning active-site groups directly involved in the chemical reaction (e.g., the catalytic triad in serine proteases), and the importance of hydrogen-bonding groups is supported by mutagenesis in many cases (e.g., refs. 1 and 3-5).In addition to hydrogen bonds, the cation-pi pair, formed between a side-chain or substrate cation and the negative electrostatic potential associated with the face of a pi system, has been widely discussed in hundreds of literature reports and has been shown to be important in both protein structure and proteinligand interactions (e.g., refs. 6 and 7). There has been much ...

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Section: Double-mutant Cycles To Test the Role Of Phe54 And Phe116 Inmentioning

confidence: 53%

Section: Double-mutant Cycles To Test the Role Of Phe54 And Phe116 Inmentioning

confidence: 87%

Use of anion–aromatic interactions to position the general base in the ketosteroid isomerase active site

Schwans¹,

Sunden²,

Lassila³

et al. 2013

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

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“…In the classic case of triosephosphate isomerase, substitution of Glu165 for Asp caused a 1000-fold decrease in specific activity, equivalent to the expected result if the base is removed completely (20). In the acyl-CoA dehydrogenases, the effect of the reverse mutation varies among the different enzymes.…”

Section: Discussionmentioning

confidence: 93%

Reductive Half-Reaction of Nitroalkane Oxidase: Effect of Mutation of the Active Site Aspartate to Glutamate^,

Valley¹,

Fitzpatrick

2003

Biochemistry

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The flavoenzyme nitroalkane oxidase catalyzes the oxidation of primary and secondary nitroalkanes to the respective aldehydes or ketones, releasing nitrite. The enzyme has recently been identified as being homologous to the acyl-CoA dehydrogenase family of enzymes [Daubner, S. C., Gadda, G., Valley, M. P., and Fitzpatrick, P. F. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 2702-2707. The glutamate which acts as an active site base in that family of enzymes aligns with Asp402 of nitroalkane oxidase. To evaluate the identification of Asp402 as an active site base, the effect of mutation of Asp402 to glutamate on the rate of cleavage of the nitroalkane C-H bond has been determined. Deuterium kinetic isotope effects on steady state kinetic parameters and direct measurement of the rate of flavin reduction establish that the mutation increases the ΔG ‡ for C-H bond cleavage by 1.6-1.9 kcal/mol. There is no effect on the rate of reaction of the reduced enzyme with oxygen. These results support the assignment of Asp402 as the active site base in nitroalkane oxidase.The flavoenzyme nitroalkane oxidase (NAO) 1 catalyzes the oxidation of nitroalkanes to the respective aldehydes or ketones with consumption of oxygen and release of nitrite and hydrogen peroxide (Scheme 1) (1). While several other flavoprotein oxidases are able to oxidize nitroalkanes, they require the preformed anion as a substrate in a clearly nonphysiological reaction (2). NAO is unique in that it uses the neutral forms of nitroalkanes as substrates (3); moreover, the enzyme is induced by growth of Fusarium oxysporum on nitroethane, consistent with nitroalkane oxidation being the physiological role of the enzyme (1). NAO can oxidize a broad range of primary and secondary nitroalkanes, although primary aliphatic nitroalkanes are the best substrates (4).As is the case with many flavoproteins, the reaction of NAO can be divided into reductive and oxidative half-reactions (5). In the reductive half-reaction, the nitroalkane substrate reacts with the oxidized flavin to generate the oxidized product and reduced flavin. The mechanism of this half-reaction (Scheme 2) has been proposed to involve removal of the substrate α-hydrogen as a proton, followed by attack of the resulting carbanion on the flavin (6). Loss of nitrite from the resulting adduct would generate an electrophilic species to which hydroxide adds (path a); this flavin adduct then decomposes to form reduced flavin and aldehyde or ketone. Evidence for this mechanism comes from the demonstration that an inactive 5-nitrobutyl-FAD adduct

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“…The conservation of the N-terminal hinge may be due to the consideration that the active site base is glutamate-165 (Raines et al, 1986), i.e., adjacent to the hinge.…”

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confidence: 99%

Determination of the amino acid requirements for a protein hinge in triosephosphate isomerase

Sun

Sampson

1998

Protein Science

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We have determined the sequence requirements for a protein hinge in triosephosphate isomerase. The codons encoding the hinge at the C-terminus of the active-site lid of triosephosphate isomerase were replaced with a genetic library of all possible 8,000 amino acid combinations. The most active of these 8,000 mutants were selected using in vivo complementation of a triosephosphate isomerase deficient strain of E. coli, DF502. Approximately 3% of the mutants complement DF502 with an activity that is above 70% of wild-type activity. The sequences of these hinge mutants reveal that the solutions to the hinge flexibility problem are varied. Moreover, these preferences are sequence dependent; that is, certain pairs occur frequently. They fall into six families of similar sequences. In addition to the hinge sequences expected on the basis of phylogenetic analysis, we selected three new families of 3-amino-acid hinges: X(A/S)(L/K/ M), X(aromatic/P-branched)(L/K), and XP(S/N). The absence of these hinge families in the more than 60 known species of triosephosphate isomerase suggests that during evolution, not all of sequence space is sampled, perhaps because there is no neutral mutation pathway to access the other families.

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Reaction energetics of a mutant triose phosphate isomerase in which the active-site glutamate has been changed to aspartate

Cited by 118 publications

References 36 publications

Use of anion–aromatic interactions to position the general base in the ketosteroid isomerase active site

Use of anion–aromatic interactions to position the general base in the ketosteroid isomerase active site

Reductive Half-Reaction of Nitroalkane Oxidase: Effect of Mutation of the Active Site Aspartate to Glutamate^,

Determination of the amino acid requirements for a protein hinge in triosephosphate isomerase

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Reaction energetics of a mutant triose phosphate isomerase in which the active-site glutamate has been changed to aspartate

Cited by 118 publications

References 36 publications

Use of anion–aromatic interactions to position the general base in the ketosteroid isomerase active site

Use of anion–aromatic interactions to position the general base in the ketosteroid isomerase active site

Reductive Half-Reaction of Nitroalkane Oxidase: Effect of Mutation of the Active Site Aspartate to Glutamate,

Determination of the amino acid requirements for a protein hinge in triosephosphate isomerase

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Reductive Half-Reaction of Nitroalkane Oxidase: Effect of Mutation of the Active Site Aspartate to Glutamate^,