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

The 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 electron-withdrawing effect of K+ may help to polarise the aromatic ring in order to aid the hydride-transfer

New type of interaction between the SARAH domain of the tumour suppressor RASSF1A and its mitotic kinase Aurora A

Abstract The tumour suppressor protein RASSF1A is phosphorylated by Aurora A kinase, thereby impairing its tumour suppressor function. Consequently, inhibiting the interaction between Aurora A and RASSF1A may be used for anti-tumour therapy. We used recombinant variants of RASSF1A to map the sites of interaction with Aurora A. The phosphorylation kinetics of three truncated RASSF1A variants has been analysed. Compared to the RASSF1A form lacking the 120 residue long N-terminal part, the K m value of the phosphorylation is increased from 10 to 45 μM upon additional deletion of the C-terminal SARAH domain. On the other hand, deletion of the flexible loop (Δ177–197) that precedes the phosphorylation site/s (T202/S203) results in a reduction of the k cat value from about 40 to 7 min−1. Direct physical interaction between the isolated SARAH domain and Aurora A was revealed by SPR. These data demonstrate that the SARAH domain of RASSF1A is involved in the binding to Aurora A kinase. Structural modelling confirms that a novel complex is feasible between the SARAH domain and the kinase domain of Aurora A. In addition, a regulatory role of the loop in the catalytic phosphorylation reaction has been demonstrated both experimentally and by structural modelling

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

The key active site residues K185, Y139, D217, D241, D245, and N102 of Thermus thermophilus 3 isopropylmalate dehydrogenase Tt IPMDH have been replaced, one by one, with Ala. A drastic decrease in the kcat value 0.06 compared to that of the wild type enzyme has been observed for the K185A and D241A mutants. Similarly, the catalytic interactions Km values of these two mutants with the substrate IPM are weakened by more than 1 order of magnitude. The other mutants retained some 1 amp; 8722;13 of the catalytic activity of the wild type enzyme and do not exhibit appreciable changes in the substrate Km values. The pH dependence of the wild type enzyme activity pK 7.4 is shifted toward higher values for mutants K185A and D241A pK values of 8.4 and 8.5, respectively . For the other mutants, smaller changes have been observed. Consequently, K185 and D241 may constitute a proton relay system that can assist in the abstraction of a proton from the OH group of IPM during catalysis. Molecular dynamics simulations provide strong support for the neutral character of K185 in the resting state of the enzyme, which implies that K185 abstracts the proton from the substrate and D241 assists the process via electrostatic interactions with K185. Quantum mechanics molecular mechanics calculations revealed a significant increase in the activation energy of the hydride transfer of the redox step for both D217A and D241A mutants. Crystal structure analysis of the molecular contacts of the investigated residues in the enzyme amp; 8722;substrate complex revealed their additional importance in particular that of K185, D217, and D241 in stabilizing the domain closed active conformation. In accordance with this, small angle X ray scattering measurements indicated the complete absence of domain closure in the cases of D217A and D241A mutants, while only partial domain closure could be detected for the other mutants. This suggests that the same residues that are important for catalysis are also essential for inducing domain closur