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Structural and kinetic evidence for an extended hydrogen-bonding network in catalysis of methyl group transfer. Role of an active site asparagine residue in activation of methyl transfer by methyltransferases.

ABSTRACT: The methyltetrahydrofolate (CH(3)-H(4)folate) corrinoid-iron-sulfur protein (CFeSP) methyltransferase (MeTr) catalyzes transfer of the methyl group of CH(3)-H(4)folate to cob(I)amide. This key step in anaerobic CO and CO(2) fixation is similar to the first half-reaction in the mechanisms of other cobalamin-dependent methyltransferases. Methyl transfer requires electrophilic activation of the methyl group of CH(3)-H(4)folate, which includes proton transfer to the N5 group of the pterin ring and poises the methyl group for reaction with the Co(I) nucleophile. The structure of the binary CH(3)-H(4)folate/MeTr complex (revealed here) lacks any obvious proton donor near the N5 group. Instead, an Asn residue and water molecules are found within H-bonding distance of N5. Structural and kinetic experiments described here are consistent with the involvement of an extended H-bonding network in proton transfer to N5 of the folate that includes an Asn (Asn-199 in MeTr), a conserved Asp (Asp-160), and a water molecule. This situation is reminiscent of purine nucleoside phosphorylase, which involves protonation of the purine N7 in the transition state and is accomplished by an extended H-bond network that includes water molecules, a Glu residue, and an Asn residue (Kicska, G. A., Tyler, P. C., Evans, G. B., Furneaux, R. H., Shi, W., Fedorov, A., Lewandowicz, A., Cahill, S. M., Almo, S. C., and Schramm, V. L. (2002) Biochemistry 41, 14489-14498). In MeTr, the Asn residue swings from a distant position to within H-bonding distance of the N5 atom upon CH(3)-H(4)folate binding. An N199A variant exhibits only approximately 20-fold weakened affinity for CH(3)-H(4)folate but a much more marked 20,000-40,000-fold effect on catalysis, suggesting that Asn-199 plays an important role in stabilizing a transition state or high energy intermediate for methyl transfer.

SUBMITTER: Doukov TI

PROVIDER: S-EPMC3966722 | biostudies-literature | 2007 Mar

REPOSITORIES: biostudies-literature

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Structural and kinetic evidence for an extended hydrogen-bonding network in catalysis of methyl group transfer. Role of an active site asparagine residue in activation of methyl transfer by methyltransferases.

Doukov Tzanko I TI Hemmi Hisashi H Drennan Catherine L CL Ragsdale Stephen W SW

The Journal of biological chemistry 20061215 9

The methyltetrahydrofolate (CH(3)-H(4)folate) corrinoid-iron-sulfur protein (CFeSP) methyltransferase (MeTr) catalyzes transfer of the methyl group of CH(3)-H(4)folate to cob(I)amide. This key step in anaerobic CO and CO(2) fixation is similar to the first half-reaction in the mechanisms of other cobalamin-dependent methyltransferases. Methyl transfer requires electrophilic activation of the methyl group of CH(3)-H(4)folate, which includes proton transfer to the N5 group of the pterin ring and p ...[more]

PMID: 17172470

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Project description:I.r. difference spectra are presented for 3-(indol-3-yl)acryloyl-, cinnamoyl-, 3-(5-methylthien-2-yl)acryloyl-, dehydrocinnamoyl- and dihydrocinnamoyl-chymotrypsins at low pH, where the acyl-enzymes are catalytically inactive. At least two absorption bands are seen in each case in the ester carbonyl stretching region of the spectrum. Cinnamoyl-chymotrypsin substituted at the carbonyl carbon atom with 13C was prepared. A difference spectrum in which 13C-substituted acyl-enzyme was subtracted from [12C]acyl-enzyme shows two bands in the ester carbonyl region and thus confirms the assignment of the features to the single ester carbonyl group. The frequencies of the ester carbonyl bands are interpreted in terms of differential hydrogen-bonding. In each case a lower-frequency relatively narrow band is assigned to a productive potentially reactive binding mode in which the carbonyl oxygen atom is inserted in the oxyanion hole of the enzyme active centre. The higher-frequency band, which is broader, is assigned to a non-productive binding mode in each case, where a water molecule bridges from the carbonyl oxygen atom to His-57; this mode is equivalent to the crystallographically determined structure of 3-(indol-3-yl)acryloyl-chymotrypsin, i.e. the Henderson structure. A difference spectrum of dihydrocinnamoyl-chymotrypsin taken at higher pH shows resolution of a feature centred upon 1731 cm-1, which is assigned to a non-bonded conformer in which the carbonyl oxygen atom is not hydrogen-bonded. Perturbation of the protein spectrum in the presence of acyl groups is interpreted in terms of enhanced structural rigidity. It is reported that the ester carbonyl region of the difference spectrum of cinnamoyl-subtilisin is complicated by overlap of features that arise from protein perturbation. Measurements of carbonyl absorption frequencies in a number of solvents of the methyl esters of the acyl groups used to make acyl-enzymes have permitted determination of the apparent dielectric constants experienced by carbonyl groups in the enzyme active centre as well as a discussion of the effects of polarity. The ester carbonyl bond strengths of the various conformations were estimated by using simple harmonic oscillator theory and an empirical relation between the force constants and bond strengths. The fractional bond breaking induced by hydrogen-bonding was used to calculate rate enhancement factors by using absolute reaction rate theory.(ABSTRACT TRUNCATED AT 400 WORDS)

Dataset Information

Structural and kinetic evidence for an extended hydrogen-bonding network in catalysis of methyl group transfer. Role of an active site asparagine residue in activation of methyl transfer by methyltransferases.

Publications

Structural and kinetic evidence for an extended hydrogen-bonding network in catalysis of methyl group transfer. Role of an active site asparagine residue in activation of methyl transfer by methyltransferases.

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