No abstract

The homohexameric enzyme methylglyoxal synthase (MGS) converts dihydroxyacetone phosphate (DHAP) to methylglyoxal and phosphate. This enzyme is allosterically inhibited by phosphate. The allosteric signal induced by phosphate in MGS from Thermus sp. GH5 (TMGS) has been tracked by site-directed mutagenesis, from the binding site of phosphate to the pathways that transmit the signal, and finally to the active site which is the receiver of the signal. In TMGS, Ser-55 distinguishes the inhibitory phosphate from the phosphoryl group of the substrate, DHAP, and transmits the allosteric signal through Pro-82, Arg-97 and Val-101 to the active site. Furthermore, the addition of a C-terminal tail to TMGS reinforces the allosteric signal by introducing a new salt bridge between Asp-10 and an Arg in this tail. Lastly, the active site amino acid, Gly-56, is shown to be involved in both allostery and phosphate elimination step from DHAP by TMGS. Interestingly, some of the mutations also trigger homotropic allostery, supporting the hypothesis that allostery is an intrinsic property of all dynamic proteins. The details of the TMGS allosteric network discussed in this study can serve as a model system for understanding the enigmatic allosteric mechanism of other proteins.

Employment of enzyme immobilization technique for α‐amylase, provides useful approaches to improve enzyme resistance to harsh conditions and reusability. Two silica supports, Ionic Liquid‐Based Periodic Mesoporous Organosilica which we call it PMO‐IL, and SBA‐15, have been used for immobilization of α‐amylase. We found that proper electrostatic interactions result in a higher immobilization yield of PMO‐IL rather than SBA‐15. The kinetics parameters show that α‐amylase@PMO‐IL activity has slightly decreased in comparison to free enzyme and the stability studies indicate that it is even more active than free enzyme in the natural pH which shows the improving role of ionic liquid on enzyme structure and function. Also, both immobilized enzymes have 2 times more stability at 70 °C and 80 °C after 60 min in comparison to free enzyme. Finally, the recovery efficiency of α‐amylase@PMO‐IL is 88% of its initial activity after 4 cycles which is 23% more than a previous report.

An amylopullulanase (L14-APU) from an Iranian thermophilic bacterium was purified and the effect of acarbose, as a general inhibitor of α-amylases, on pullulan and starch hydrolysis catalyzed by L14-APU was investigated. The inhibition is a competitive type whereas inhibition constants for pullulan and starch are 99 µM and 72 µM, respectively. Investigation of the reaction rate in a system contains competitive substrates and the inhibition type of acarbose in presence of different substrates suggests that L14-APU possesses only one active site for two activities. The analysis of metal ions and other reagents effects has shown that Ca 2+ , Mg 2+ , Mn 2+ and Co 2+ enhanced both activities of the enzyme while N-bromosuccinimide treatment leads to the complete inactivation of the enzyme. The enzyme activity increased in the presence of low concentration of SDS as a surfactant.

Lately it has been proposed that interaction between two positively charged side chains can stabilize the folded state of proteins. To further explore this point, we studied the effect of histidine-histidine interactions on thermostability of methylglyoxal synthase from Thermus sp. GH5 (TMGS). The crystal structure of TMGS revealed that His23, Arg22, and Phe19 are in close distance and form a surface loop. Here, two modified enzymes were produced by site-directed mutagenesis (SDM); one of them, one histidine (TMGS-HH(O)), and another two histidines (TMGS-HHH(O)) were inserted between Arg22 and His23 (H(O)). In comparison with the wild type, TMGS-HH(O) thermostability increased remarkably, whereas TMGS-HHH(O) was very unstable. To explore the role of His23 in the observed phenomenon, the original His23 in TMGS-HHH(O) was replaced with Ala (TMGS-HHA). Our data showed that the half-life of TMGS-HHA decreased in relation to the wild type. However, its half-life increased in comparison with TMGS-HHH(O). These results demonstrated that histidine-histidine interactions at position 23 in TMGS-HH(O) probably have the main role in TMGS thermostability.