Attempt on Magnification of the Mechanism of Enzyme Catalyzed Reaction through Bio-geometric Model for the Five Points Circle in the Triangular Form of Lineweaver-Burk Plot

oai:sciengtexopen.org:article/120The bio-geometrical model is dealing with correlation between the “five events for enzyme catalyzed reaction” and “triple point event serving groups on the circle” in triangle obtained for the graphical presentation of the double reciprocal for magnification of the mechanism of enzyme catalyzed reaction. This model is based on the nine point circle in triangle of the double reciprocal plot. The five significant points (B, D, E, F and G) resulted for the circle with x – and y – coordinates. The present attempt is considering interactions among enzymes and substrates for the successful release of product through each and every point on the circle in triangle. The controlling role of the point, “O”, center of circle in each and every event of the biochemical reaction is obligatory.  The model is allotting specific role for the significant events in the biochemical reaction catalyzed by the enzymes. The enzymatic catalysis is supposed to be completed through five events, which may be named as, “Bio-geometrical events of enzyme catalyzed reaction”. These five events for enzyme catalyzed reaction include: (1) Initial event of enzymatic interaction with the substrates; (2) Event of the first transition state for the formation of “enzyme-substrate” complex; (3) Event of the second transition state for the formation of “enzyme-product” complex; (4) Event of release of the product and relieve enzyme and (5) The event of directing the enzyme to continue the reaction. The model utilizes the “triple point serving group on the circle” for the success of each and every event in the biochemical reaction. Thus, there is involvement of the three points including the point “O” for each event in the enzyme catalyzed reaction. The group of points serving for carrying out the event may be classified into five conic sections like: B-O-E; E-O-G; G-O-D; D-O-F and F-O-B. The bio-geometrical model is correlation between the “five events for enzyme catalyzed reaction” and “triple point event serving groups on the circle” in a triangle of the double reciprocal plot

Phosphorylation sites in BubR1 that regulate kinetochore attachment, tension, and mitotic exit

BubR1 kinase is essential for the mitotic checkpoint and also for kinetochores to establish microtubule attachments. In this study, we report that BubR1 is phosphorylated in mitosis on four residues that differ from sites recently reported to be phosphorylated by Plk1 (Elowe, S., S. Hummer, A. Uldschmid, X. Li, and E.A. Nigg. 2007. Genes Dev. 21:2205–2219; Matsumura, S., F. Toyoshima, and E. Nishida. 2007. J. Biol. Chem. 282:15217–15227). S670, the most conserved residue, is phosphorylated at kinetochores at the onset of mitosis and dephosphorylated before anaphase onset. Unlike the Plk1-dependent S676 phosphorylation, S670 phosphorylation is sensitive to microtubule attachments but not to kinetochore tension. Functionally, phosphorylation of S670 is essential for error correction and for kinetochores with end-on attachments to establish tension. Furthermore, in vitro data suggest that the phosphorylation status of BubR1 is important for checkpoint inhibition of the anaphase-promoting complex/cyclosome. Finally, RNA interference experiments show that Mps1 is a major but not the exclusive kinase that specifies BubR1 phosphorylation in vivo. The combined data suggest that BubR1 may be an effector of multiple kinases that are involved in discrete aspects of kinetochore attachments and checkpoint regulation

Degradation of Cdc25A by \u3b2-TrCP during S phase and in response to DNA damage

The Cdc25A phosphatase is essential for cell-cycle progression because of its function in dephosphorylating cyclin-dependent kinases. In response to DNA damage or stalled replication, the ATM and ATR protein kinases activate the checkpoint kinases Chk1 and Chk2, which leads to hyperphosphorylation of Cdc25A1\u20133. These events stimulate the ubiquitin-mediated pro- teolysis of Cdc25A1,4,5 and contribute to delaying cell-cycle progression, thereby preventing genomic instability1\u20137. Here we report that b-TrCP is the F-box protein that targets phosphory- lated Cdc25A for degradation by the Skp1/Cul1/F-box protein complex. Downregulation of b-TrCP1 and b-TrCP2 expression by short interfering RNAs causes an accumulation of Cdc25A in cells progressing through S phase and prevents the degradation of Cdc25A induced by ionizing radiation, indicating that b-TrCP may function in the intra-S-phase checkpoint. Consistent with this hypothesis, suppression of b-TrCP expression results in radioresistant DNA synthesis in response to DNA damage\u2014a phenotype indicative of a defect in the intra-S-phase checkpoint that is associated with an inability to regulate Cdc25A properly. Our results show that b-TrCP has a crucial role in mediating the response to DNA damage through Cdc25A degradation

Science as an Adventure - Lessons for the Young Scientist

I graduated as MD in 1965, after gaining substantial exposure to basic science in the course of my medical studies. I obtained my PhD degree in 1969, and thus, in essence, I have been actively engaged in conducting research for over half a century. It goes without saying, that to be successful in any field one has to love what one does. This is most important in experimental science, since experiments do not always work and at times the results disprove what you had been hoping for. Moments when everything comes together and you can shout “Eureka” are few and far between, and the outcomes attained might not exactly fit the starting hypothesis. However, such unexpected results can turn out to be of greatest importance. In one of my first experiments during my postdoctorate period, I observed quite by accident that the enzyme involved in the degradation of the protein tyrosine aminotransferase required energy. I wondered why would the degradation of an intracellular protein require energy, whereas to our knowledge protein degradation outside of cells – e.g. the digestion of food – does not. This “accidental” observation led me to assume the existence of some kind of novel, unknown energy-dependent mechanism that governs highly selective protein degradation within cells. I was very impressed by this finding, and all my subsequent work was influenced by this one experiment. Although it had been reached fortuitously, I considered the observation to be important. It might have been mere luck that I chose to do this type of experiment early on in my career, but luck by itself would not have steered me toward further achievements. I had to embark on serious scientific work to pursue this unique finding – and I have been pursuing it ever since

Roles of the anaphase-promoting complex/cyclosome and of its activator Cdc20 in functional substrate binding

The anaphase-promoting complex/cyclosome (APC/C) is a multisubunit ubiquitin-protein ligase that targets for degradation cell-cycle regulatory proteins during exit from mitosis and in the G(1) phase of the cell cycle. The activity of APC/C in mitosis and in G(1) requires interaction with the activator proteins Cdc20 and Cdh1, respectively. Substrates of APC/C–Cdc20 contain a recognition motif called the “destruction box” (D-box). The mode of the action of APC/C activators and their possible role in substrate binding remain poorly understood. Several investigators suggested that Cdc20 and Cdh1 mediate substrate recognition, whereas others proposed that substrates bind to APC/C or to APC/C–activator complexes. All these studies used binding assays, which do not necessarily indicate that substrate binding is functional and leads to product formation. In the present investigation we examined this problem by an “isotope-trapping” approach that directly demonstrates productive substrate binding. With this method we found that the simultaneous presence of both APC/C and Cdc20 is required for functional substrate binding. By contrast, with conventional binding assays we found that either Cdc20 or APC/C can bind substrate by itself, but only at low affinity and relaxed selectivity for D-box. Our results are consistent with models in which interaction of substrate with specific binding sites on both APC/C and Cdc20 is involved in selective and productive substrate binding

The ubiquitin system

It has been often stated that until recently the ubiquitin system was thought to be mainly a 'garbage disposal' for the removal of abnormal or damaged proteins. This statement is certainly not true for those who have been interested in the selective and regulated degradation of proteins in cells. The dynamic turnover of cellular proteins was discovered in the pioneering studies of Rudolf Schoenheimer in the 1930s, when he first used isotopically labeled compounds for biological studies. Between 1960 and1970 it became evident that protein degradation in animal cells is highly selective, and is important in the control of specific enzyme concentrations. The molecular mechanisms responsible for this process, however, remained unknown. Some imaginative models have been proposed to account for the selectivity of protein degradation, such as one suggesting that all cellular proteins are rapidly engulfed into the lysosome, but only short-lived proteins are degraded in the lysosome, whereas long-lived proteins escape back to the cytosol