In this study, we attempted to understand the mechanism of regulation of the activity and allosteric behavior of the pyruvate kinase M 2 enzyme and two of its missense mutations, H391Y and K422R, found in cells from Bloom syndrome patients, prone to develop cancer. Results show that despite the presence of mutations in the intersubunit contact domain, the K422R and H391Y mutant proteins maintained their homotetrameric structure, similar to the wild-type protein, but showed a loss of activity of 75 and 20%, respectively. Interestingly, H391Y showed a 6-fold increase in affinity for its substrate phosphoenolpyruvate and behaved like a non-allosteric protein with compromised cooperative binding. However, the affinity for phosphoenolpyruvate was lost significantly in K422R. Unlike K422R, H391Y showed enhanced thermal stability, stability over a range of pH values, a lesser effect of the allosteric inhibitor Phe, and resistance toward structural alteration upon binding of the activator (fructose 1,6-bisphosphate) and inhibitor (Phe). Both mutants showed a slight shift in the pH optimum from 7.4 to 7.0. Although this study signifies the importance of conserved amino acid residues in long-range communications between the subunits of multimeric proteins, the altered behavior of mutants is suggestive of their probable role in tumor-promoting growth and metabolism in Bloom syndrome patients with defective pyruvate kinase M 2 .Pyruvate kinase (PK 3 ; EC 2.7.1.40), a pacemaker of the glycolytic pathway, catalyzes irreversibly the transphosphorylation from P-enolpyruvate to ADP, generating pyruvate and ATP (1, 2). There are four different isozymes (L, R, M 1 , and M 2 ) in mammalian tissues, which differ in their regulatory properties. These isozymes are allosteric in nature with the exception of the M 1 form, present in skeletal muscle and brain (3-7). PKM 2 is a ubiquitous prototype enzyme present in all tissues during the embryonic stage and is gradually replaced by other isozymic forms in specific tissues during development. The M 2 , L, and R isozymes show homotropic cooperative activation with P-enolpyruvate and heterotropic cooperative activation with Fru-1,6-P 2 (8 -10). The M 1 isozyme is regulated by neither P-enolpyruvate nor Fru-1,6-P 2 because of its intrinsic active conformation in the R-state (5, 6). Under unfavorable conditions such as hypoxia and lack of glucose supply, the anaerobic tissues and tumor cells rely heavily on PKM 2 for ATP production (7). Therefore, stringent control of PK activity is of great importance not only for cell metabolism but also for tumorigenic proliferation.The M 1 and M 2 isozymes are produced from a single gene locus by mutually exclusive alternative splicing (11)(12)(13)(14). In the human M 1 and M 2 isozymes, the exon that is exchanged because of alternative splicing encodes 56 amino acids, in which a total of 22 amino acids differ within a length of 45 residues. The residues located in this region form the major intersubunit contact domain (8). The distinguishable kinetic pro...

The crystal structure of a bifunctional inhibitor of -amylase and trypsin (RATI) from ragi seeds (Indian ®nger millet, Eleusine coracana Gaertneri) has been determined by X-ray diffraction at 2.2 A Ê resolution. The inhibitor consists of 122 amino acids, with ®ve disul®de bridges, and belongs to the plant -amylase/trypsin inhibitor family. The crystals were grown by the microdialysis method using ammonium sulfate as a precipitating agent. The structure was determined by the molecular-replacement method using as models the structures of Corn Hageman factor inhibitor (CHFI) and of RATI at 2.9 A Ê resolution determined previously. It has been re®ned to an R factor of 21.9%. The structure shows an r.m.s. deviation for C atoms of 2.0 A Ê compared with its own NMR structure, whereas the corresponding value compared with CHFI is found to be 1.4 A Ê . The r.m.s. difference for C atoms when compared with the same protein in the structure of the complex with -amylase is 0.7 A Ê . The conformations of trypsin-binding loop and the -amylase-binding N-terminal region were also found to be similar in the crystal structures of native RATI and its complex with -amylase. These regions differed considerably in the NMR structure.

Calcium plays a pivotal role in the pathogenesis of amoebiasis, a major disease caused by Entamoeba histolytica. Several EF-hand containing calcium-binding proteins (CaBPs) have been identified from E. histolytica. Even though these proteins have very high sequence similarity, they bind to different target proteins in a Ca2+ dependent manner, leading to different functional pathways (Yadava et al., Mol Biochem Parasito 1997;84:69-82; Chakrabarty et al., J Biol Chem 2004;279:12898-12908) The crystal structure of the Entamoeba histolytica calcium binding protein-1 (EhCaBP1) has been determined at 2.4 A resolution. The crystals were grown using MPD as precipitant and they belong to P6(3) space group with unit cell parameters of a = 95.25 A, b = 95.25 A, c = 64.99 A. Only two out of the four expected EF hand motifs could be modeled into the electron density map and the final model refined to R factor of 25.6% and Free_R of 28%. Unlike CaM, the first two EF hand motifs in EhCaBP1 are connected by a long helix and form a dumbbell shaped structure. Owing to domain swapping oligomerization three EhCaBP1 molecules interact in a head to tail manner to form a triangular trimer. This arrangement allows the EF-hand motif of one molecule to interact with that of an adjacent molecule to form a two EF-hand domain similar to that seen in the N-terminal domain of the NMR structure of CaBP1, calmodulin and troponin C. The oligomeric state of EhCaBP1 results in reduced flexibility between domains and may be responsible for the more limited set of targets recognized by EhCaBP1.

Cysteine plays a major role in the antioxidative defense mechanisms of the human parasite Entameoba histolytica. The major route of cysteine biosynthesis in this parasite is the condensation of O-acetylserine with sulfide by the de novo cysteine biosynthetic pathway involving two key enzymes O-acetyl-L-serine sulfhydrylase (OASS) and serine acetyl transferase (SAT). The crystal structure of native OASS from Entameoba histolytica (EhOASS) has been determined at 1.86 A resolution and in complex with its product cysteine at 2.4 A resolution. In comparison with other known OASS structures, insertion in the N-terminal region and C-terminal helix reveal critical differences, which may influence the protein-protein interactions. In spite of lacking chloride binding site at the dimeric interface, the N-terminal extension compared with other known cysteine synthases, participates in dimeric interactions in an interesting domain swapping manner, enabling it to form a stronger dimer. Sulfate is bound in the active site of the native structure, which is replaced by cysteine in the cysteine bound form causing reorientation of the small N-terminal domain and thus closure of the active site. Ligand binding constants of OAS, Cys, and Met with EhOASS are comparable with other known OASS indicating similar active site arrangement and dynamics. The cysteine complexed structure represents the snapshot of the enzyme just before releasing the final product with a closed active site. The C-terminal helix positioning in the EhOASS may effect its interactions with EhSAT and thus influencing the formation of the cysteine synthase complex in this organism.

Human seminal plasma contains a large array of proteins required for the normal physiology and metabolism of spermatozoa. These are mainly secreted from prostate epithelium, testes, and seminal vesicles. Fortunately, many of these are found to be present at elevated concentration in seminal plasma and act as a biomarker of different carcinomas as their levels are also enhanced in serum and are found to be involved in tumor progression and metastasis apart from fertility. The proteins which were overexpressed in the seminal plasma of prostate carcinoma patients were identified by 2-DE and MALDI-TOF/MS. We have designed a strategy to purify these four proteins prostate specific antigen (PSA), prostatic acid phosphatase (PAP), Zinc alpha2-glycoprotein (ZAG), and progastricsin (PG), together in homogeneity by using simple chromatographic techniques. Acidic and basic fractions of human seminal plasma were separated by ion exchange chromatography and further purified by gel permeation chromatography. Our results form a new and valuable resource for those attempting structure-based drug designing for prostate and other cancers where the amount of proteins is required in plenty and in native form.