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Research has been conducted on the creatine kinase which catalyzes creatine reversible conversion and adenosine triphosphate to phosphocreatine and adenosine diphosphate. The X-ray structure of creatine kinase bound to a transition-state analogue complex has been investigated and the details are reported.

Research has been conducted on the human muscle creatine kinase. The use of the site-directed mutagenesis in replacing the Cys282 of the human muscle creatine kinase with serine and methionine is discussed.

The guanidine substrate creatine undergoes reversible phosphorylation catalyzed by the creatine kinase and magnesium-adenosine triphosphate. Crystallographic analysis and site-directed mutagenesis of the enzyme identify Glu232 as the likely catalytic residue and Asp326 aiding in the proper assignment of substrates for catalysis.

Previous research has suggested that a large conformational change is required in the catalysis by the flavoenzyme thioredoxin reductase (TrR) of the reduction of the small redox protein thioredoxin (Tr). To test this hypothesis, a complex between TrR and its substrate Tr is studied. Results indicate that the complex is a stable mixed disulfide whose lack of an electron path from FADH2 to the disulfide in the FR conformation accounts for its lack of thioredoxin reduction activity.

The four modified forms of thioredoxin reductase (TrxR) from Plasmodiym falciparum were compared with the wild-type enzyme. The results of reductive titration and pre-steady kinetic studies showed that the C-terminal cysteines are in communication with the active site nascent dithiol. It was shown that both Cys535 and Cys540 can accept the reducing equivalents passed from the active site dithiol.

A study was conducted to clone wild-type Mycobacterium leprae thioredoxin reductase-thioredoxin fusion enzyme by NADPH. Protein expression in Escherichia coli was determined by adding an isopropyl beta-thiogalactopyranoside solution to cultures in mid-log growth phase. Results suggested that a linked thioredoxin may be utilized as a probe for analyzing conformation changes. Findings also showed that M leprae TrxR-Trx supported slower reaction rates than E coli TrxR.

Creatine kinase (CK) catalyzes the reversible conversion of creatine and ATP to phosphocreatine and ADP, thereby helping maintain energy homeostasis in the cell. Here we report the first X-ray structure of CK bound to a transition-state analogue complex (CK-TSAC). Cocrystallization of the enzyme from Torpedo californica (TcCK) with ADP-Mg(2+), nitrate, and creatine yielded a homodimer, one monomer of which was liganded to a TSAC complex while the second monomer was bound to ADP-Mg(2+) alone. The structures of both monomers were determined to 2.1 A resolution. The creatine is located with the guanidino nitrogen cis to the methyl group positioned to perform in-line attack at the gamma-phosphate of ATP-Mg(2+), while the ADP-Mg(2+) is in a conformation similar to that found in the TSAC-bound structure of the homologue arginine kinase (AK). Three ligands to Mg(2+) are contributed by ADP and nitrate and three by ordered water molecules. The most striking difference between the substrate-bound and TSAC-bound structures is the movement of two loops, comprising residues 60-70 and residues 323-332. In the TSAC-bound structure, both loops move into the active site, resulting in the positioning of two hydrophobic residues (one from each loop), Ile69 and Val325, near the methyl group of creatine. This apparently provides a specificity pocket for optimal creatine binding as this interaction is missing in the AK structure. In addition, the active site of the transition-state analogue complex is completely occluded from solvent, unlike the ADP-Mg(2+)-bound monomer and the unliganded structures reported previously.