Glutamate 270 plays an essential role in K⁺‐activation and domain closure of Thermus thermophilus isopropylmalate dehydrogenase

Abstract

Edited by Peter Brzezinski Keywords:Isopropylmalate dehydrogenase Activation by K + Site-directed mutagenesis X-ray crystallography Small angle X-ray scattering Fluorescence resonance energy transfer a b s t r a c tThe mutant E270A of Thermus thermophilus 3-isopropylmalate dehydrogenase exhibits largely reduced ($1%) catalytic activity and negligible activation by K + compared to the wild-type enzyme. A 3-4 kcal/mol increase in the activation energy of the catalysed reaction upon this mutation could also be predicted by QM/MM calculations. In the X-ray structure of the E270A mutant a water molecule was observed to take the place of K + . SAXS and FRET experiments revealed the essential role of E270 in stabilisation of the active domain-closed conformation of the enzyme. In addition, E270 seems to position K + into close proximity of the nicotinamide ring of NAD + and the electronwithdrawing effect of K + may help to polarise the aromatic ring in order to aid the hydride-transfer.

Dual Role of the Active Site Residues of Thermus thermophilus 3-Isopropylmalate Dehydrogenase: Chemical Catalysis and Domain Closure

“…37,57,62 c Determined previously. 57 , which 355 is similar to the observations by Miyazaki and Oshima that were interpreted as an effect of some undefined local conformational 357 changes in the NAD + binding site of this mutant. 52 It may be 358 notable that previously we have detected a significant increase…”

“…This was supported by our previous SAXS measurements, 37 although an exceptional case was also discovered. 57 Because the native gel electrophoresis experiment and the near-UV CD spectral changes observed upon the present The complete absence of FRET spectra could be observed only in cases of the last two Asp mutants (D241 and D217′). These two aspartates are interacting directly with both the catalytic Mn 2+ and the catalytic side-chain K185′ (cf.…”

“…This was supported by our previous SAXS measurements, 37 although an exceptional case was also discovered. 57 Because the native gel electrophoresis experiment and the near-UV CD spectral changes observed upon the present mutations (cf. above) raised the possibilities of conformational changes upon mutations, we tested formation of the FRET with all the active site mutants of IPMDH mentioned above.…”

“…The p K values were determined from the pH dependences of the activities by fitting the values to eq . The increase in activation energy compared to the wild-type enzyme [ΔΔ E ⧧ = − RT ln k cat (mutant)/ k cat (wild)] was estimated from the k cat values using the Arrhenius equation and is given in kilocalories per mole. b Determined previously. ,, c Determined previously d Not determined. …”

“… a The R g values were computed by two alternative methods, using the program GNOM and Guinier approximation. b Published previously c Values of the high-resolution models as retrieved from PDB entry 4F7I for the closed crystal structure of the Tt- IPMDH–Mn 2+ –IPM–NADH quaternary complex and PDB entry 2Y3Z for the open crystal structure of apo Tt -IPMDH. d The minimum values of discrepancy (in bold) indicate the best correlation between SAXS data and crystallographic model. e The fits for open/closed mixture were obtained with OLIGOMER. f V open and V closed correspond to the volume fractions of each state found by OLIGOMER. g The small variations in the calculated molecular mass are due to different numbers of residues resolved in different crystal structures. …”