http://inspirehep.net/record/499969/files/arXiv:hep-th_9905111.pdf

Large N Field Theories, String Theory and Gravity

TAXOL (Fig. 1) was isolated from the plant Taxus brevifolia (western yew) by Wani et al., who reported that the molecule has antitumour activity in several experimental systems1. In our laboratory we have found that taxol, a low molecular weight neutral compound, completely inhibits division of exponentially growing HeLa cells at low concentrations of drug (0.25 µM) that have no significant effects on DNA, RNA or protein synthesis during a 4-h incubation with the cells. HeLa cells incubated with taxol for 20 h are blocked in late G2 and/or M (ref. 2). We report here that taxol acts as a promoter of calf brain microtubule assembly in vitro, in contrast to plant products such as colchicine and podophyllotoxin, which inhibit assembly. Taxol decreases the lag time for microtubule assembly and shifts the equilibrium for assembly in favour of the microtubule, thereby decreasing the critical concentration of tubulin required for assembly. Microtubules polymerised in the presence of taxol are resistant to depolymerisation by cold (4 °C) and CaCl2 (4 mM).

Promotion of microtubule assembly in vitro by taxol

Brownian motors: noisy transport far from equilibrium

Progression of the cell cycle and control of apoptosis (programmed cell death) are thought to be intimately linked processes, acting to preserve homeostasis and developmental morphogenesis. Although proteins that regulate apoptosis have been implicated in restraining cell-cycle entry and controlling ploidy (chromosome number), the effector molecules at the interface between cell proliferation and cell survival have remained elusive. Here we show that a new inhibitor of apoptosis (IAP) protein, survivin, is expressed in the G2/M phase of the cell cycle in a cycle-regulated manner. At the beginning of mitosis, survivin associates with microtubules of the mitotic spindle in a specific and saturable reaction that is regulated by microtubule dynamics. Disruption of survivin-microtubule interactions results in loss of survivin's anti-apoptosis function and increased caspase-3 activity, a mechanism involved in cell death, during mitosis. These results indicate that survivin may counteract a default induction of apoptosis in G2/M phase. The overexpression of survivin in cancer may overcome this apoptotic checkpoint and favour aberrant progression of transformed cells through mitosis.

Control of apoptosis and mitotic spindle checkpoint by survivin.

Identification of a novel force-generating protein, kinesin, involved in microtubule-based motility

Nearly 100 years ago Michaelis and Menten published their now classic paper (Michaelis, L., and Menten, M. L. (1913) Die Kinetik der Invertinwirkung. Biochem. Z. 49, 333−369) in which they showed that the rate of an enzyme- catalyzed reaction is proportional to the concentration of the enzyme−substrate complex predicted by the Michaelis− Menten equation. Because the original text was written in German yet is often quoted by English-speaking authors, we undertook a complete translation of the 1913 publication, which we provide as Supporting Information. Here we introduce the translation, describe the historical context of the work, and show a new analysis of the original data. In doing so, we uncovered several surprises that reveal an interesting glimpse into the early history of enzymology. In particular, our reanalysis of Michaelis and Menten's data using modern computational methods revealed an unanticipated rigor and precision in the original publication and uncovered a sophisticated, comprehensive analysis that has been overlooked in the century since their work was published. Michaelis and Menten not only analyzed initial velocity measurements but also fit their full time course data to the integrated form of the rate equations, including product inhibition, and derived a single global constant to represent all of their data. That constant was not the Michaelis constant, but rather Vmax/Km, the specificity constant times the enzyme concentration (kcat/Km × E0).

/pdf/the-original-michaelis-constant-translation-of-the-1913-544uvxlwef.pdf

The Original Michaelis Constant: Translation of the 1913 Michaelis–Menten Paper

The fidelity of DNA polymerases is largely attributable to a two-step nucleotide binding mechanism. In the first step, binding contacts are initially made between the template and the incoming dNTP. The selectivity of this ground-state binding is similar in magnitude to the selectivity seen in forming base pairs in solution. In the second step, a change in protein conformation occurs, which leads to rapid incorporation of the dNTP into the growing polymer. This conformational change appears to occur globally in that it is inhibited by mismatches in the dNTP or in any of the three terminal base pairs of the primer/template. The open conformation allows rapid binding of the dNTP from solution, while the closed conformation provides steric checks for the proper Watson-Crick base pair geometry. This conformational change accounts for the extraordinary fidelity of polymerization and also provides selectivity to the exonuclease by inhibiting polymerization over a mismatch in the primer/template. The overall fidelity approaches one error in 10(10) by a combination of selectivity in polymerization (10(5)-10(6)) and in proofreading (10(3)-10(4)). This paradigm provides the theoretical basis for further investigation of the structural basis for fidelity by pointing to the essential elements of the polymerization reaction that need to be examined in order to evaluate active-site-directed mutants of polymerases to test appropriate structure/function relationships.

Conformational Coupling in DNA Polymerase Fidelity

The mechanism of inhibition of HIV-1 reverse transcriptase by three nonnucleoside inhibitors is described. Nevirapine, O-TIBO, and CI-TIBO each bind to a hydrophobic pocket in the enzyme-DNA complex close to the active site catalytic residues. Pre-steady-state kinetic analysis was used to establish the mechanism of inhibition by these noncompetitive inhibitors. Analysis of the pre-steady-state burst of DNA polymerization indicated that inhibitors blocked the chemical reaction, but did not interfere with nucleotide binding or the nucleotide-induced conformational change. Rather, in the presence of saturating concentrations of the inhibitors, the nucleoside triphosphate bound tightly (Kd, 100 nM), but nonproductively. The data suggest that an inhibitor combining the functionalities of a nonnucleoside inhibitor and a nucleotide analog could bind very tightly and specifically to reverse transcriptase and could be effective in the treatment of AIDS.

https://science.sciencemag.org/content/sci/267/5200/988.full.pdf

Mechanism of inhibition of HIV-1 reverse transcriptase by nonnucleoside inhibitors

The elementary steps of DNA polymerization catalyzed by T7 DNA polymerase have been resolved by transient-state analysis of single nucleotide incorporation, leading to the complete pathway: where E, D, N, and P represent T7 DNA polymerase, DNA primer/template, deoxynucleoside triphosphate, and inorganic pyrophosphate, respectively. A DNA primer/template consisting of a synthetic 25/36-mer has been used as a substrate for correct nucleotide incorporation of dTTP in all the experiments. The rate constants and equilibrium constants of each step have been established by direct measurement of individual reactions and fit by computer simulation of the data to obtain a single set of rate constants accounting for all the data. Analysis of the single-turnover kinetics provided measurements of equilibrium dissociation constants for 25/36-mer, dTTP, and PPI equal to 18 nM (koff/ko,), 18 FM (kI/kl), and 2 mM (k5/k5), respectively. The rate-limiting step during single-nucleotide incorporation has been identified as a con- formational change, E.D,-N - E'-D,-N, which occurs at a rate of 300 s-l (k2) upon binding of the correct dNTP. Accordingly, tighter binding of the transition states for the reaction resulting from the conformational change facilitates the phosphodiester bond formation. The chemical step itself was excluded as the rate- limiting step because of the small phosphothioate elemental effect. An observed rate constant of 70 s-l for dTTP(aS) incorporation suggest that the chemical step (k,) occurs at a fast rate, 29000 s-I. Following chemistry, the resulting ternary complex, E'.D,+l-P, undergoes a second conformational change at a rate of 1200 s-l (k4), leading to release of PPI and translocation of the DNA to continue subsequent cycles of polymerization. The rate constants of the reverse steps, 100 (k2), 118000 s-l (k3) and 18 s-l (k4), were derived as fits to the data based upon simulation of single-turnover kinetics of pyrophosphorolysis including measurements of pyrophosphate exchange and the overall equilibrium constant of 1 .O X lo4 for elongation of E-25/36-mer to E-26/36-mer and analysis of the kinetics of the pulse-chase experiment. These studies provide the first complete and self-consistent thermodynamic descriptions of DNA polymerase and establish the basis for quantitative assessment of the reactions contributing to its extraordinary fidelity. The rate constants of the reverse reaction (pyrophosphorolysis) could only be measured by using an exo- nuclease-deficient mutant of gene 5 protein because of the high exonuclease activity of the wild-type enzyme. Thc double mutation D5A,E7A resulted in complete inactivation of the exonuclease activity. The mutant was fully characterized, and all rate constants for the polymerization reaction were shown to be identical with those of the wild-type enzyme. Its excision rate for single-stranded DNA was shown to be reduced by a factor of IO6, while its excision rate for double-stranded DNA was not measurable.

Kenneth A. Johnson

Papers

The Original Michaelis Constant: Translation of the 1913 Michaelis–Menten Paper

Conformational Coupling in DNA Polymerase Fidelity

Mechanism of inhibition of HIV-1 reverse transcriptase by nonnucleoside inhibitors

Pre-steady-state kinetic analysis of processive DNA replication including complete characterization of an exonuclease-deficient mutant.

Global Kinetic Explorer: A new computer program for dynamic simulation and fitting of kinetic data