Essential role of the metal-ion in the IPM-assisted domain closure of 3-isopropylmalate dehydrogenase

Abstract

Edited by Stuart FergusonKeywords: Domain movement Substrate and metal-effect 3-Isopropylmalate dehydrogenase Small-angle X-ray scattering a b s t r a c t X-ray structures of 3-isopropylmalate dehydrogenase (IPMDH) do not provide sufficient information on the role of the metal-ion in the metal-IPM assisted domain closure. Here solution studies were carried out to test its importance. Small-angle X-ray scattering (SAXS) experiments with the Thermus thermophilus enzyme (complexes with single substrates) have revealed only a very marginal (0-5%) extent of domain closure in the absence of the metal-ion. Only the metal-IPM complex, but neither the metal-ion nor the free IPM itself, is efficient in stabilizing the native protein conformation as confirmed by denaturation experiments with Escherichia coli IPMDH and by studies of the characteristic fluorescence resonance energy transfer (FRET) signal (from Trp to bound NADH) with both IPMDHs. A possible atomic level explanation of the metal-effect is given.

Transient kinetic studies reveal isomerization steps along the kinetic pathway of Thermus thermophilus 3‐isopropylmalate dehydrogenase

“…The K d values of IPM binding were determined by fitting the dependences of the rate constants of FRET signal formation and the FRET amplitudes on the concentration of IPM to a single binding hyperbola. The K d values obtained from the whole FRET signal (A 1 + A 2 ) agree well with the value determined earlier [19]. ND, not determined.…”

“…Control kinetic experiments with EDTA indicated that EDTA does not compete with binding of IPM; that is, inhibition by EDTA is solely attributable to withdrawal of the metal ion from the IPMDH active site .…”

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

“…In our previous studies with the wild‐type Tt ‐IPMDH we clearly detected domain closure in the complex with Mn 2+ and IPM [19]. This experiment was repeated with the mutant E270A that also binds the substrate MnIPM.…”

“…The radii of gyration and the scattering patterns from the crystallographic models of wild‐type apo Tt ‐IPMDH (pdb: 2Y3Z) and its substrate complex with Mn 2+ , IPM and NADH (pdb: 4F7I) were computed using the program CRYSOL [18]. The program OLIGOMER [15] was used to calculate the ratio of open‐ and closed‐form species present in Tt ‐IPMDH solutions as described in [19].…”

“…The kinetic measurements have been carried out as described in the Section 2 under the conditions specified in the legends to Fig. 2 These kinetic constants has been determined earlier [19]. The minimum values of discrepancy (in bold) indicate the best correlation between SAXS data and crystallographic model.…”