Sitting beside his lab bench, Sam reflects on his research project. He explains that he is investigating an enzyme that aids the HIV virus in its replication and multiplication to its eventual defeat of the human immune system. The enzyme is HIV reverse transcriptase (RT), the enzyme that transcribes viral RNA into DNA. If this enzyme could be shut down, then the viral load of HIV patients would be dramatically reduced. There exist a number of drugs that almost shut down RT. Examples such as AZT and ddI, both nucleoside analogs, are very successful in doing so, but the sheer number of HIV virions present in HIV-positive individuals, and the ability of retroviruses such as HIV to mutate very efficiently, cause new strains of HIV to be selected in the patient. These new strains perform the chemistry needed for HIV replication slightly differently from the "wild-type" RT. They are more efficient than the wild-type RT under the conditions created by the presence of nucleoside analogs, and thus they perpetuate the HIV condition.
The HIV virus mutates with great regularity to a small set of mutants that can operate tolerably under the influence of the drug. The differences in the chemistry between these mutants can be investigated by pre-steady-state kinetic methods. The machinery Sam uses (in addition to the usual run of polyacrylamide gels, radioactive nuclei, organic solvents, and paperwork) are the quench-flow and the stop-flow machines. They enable him to view the chemical events that take place on the millisecond time scale, either directly (stop-flow) or indirectly (quench-flow).
How the Stop-Flow and Quench-Flow Work
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Stop-flow being used to analyze the kinetic activity of the motor protein kinesin.
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Pre-steady state kinetics is simply defined as the enzyme kinetics that take place during the short time before the enzymes and reactants in a reaction are able to reach the steady state. Steady-state kinetics can be studied using the methods of Michaelis and Menten. The way quench-flow and stop-flow work is as follows: The quench-flow is a very finely tuned, computer-controlled machine that is designed to mix enzyme and reactants very rapidly to start the enzymatic reaction, and then quench it after a defined time. The reaction time currently ranges from about five milliseconds to several seconds. The quench-flow is used when the enzymatic reaction does not involve a component that can be spectrally monitored in real time. An example of this kind of reaction is the polymerization of DNA by a DNA polymerase, such as the HIV reverse transcriptase or the human mitochondrial DNA polymerase. There is no way to directly monitor this reaction, so the reaction is allowed to proceed for a short time and is quenched. The time course of the reaction can then be analyzed by electrophoretic methods.
The stop-flow method is very similar to the quench-flow method in that the stop-flow machine is designed to mix the enzyme and reactants very rapidly to start the reaction. But the advantage of the stop-flow over the quench-flow is that it has a spectrophotometric detection system hooked up to it so that the reaction can be monitored in real time. An example is a case where one is measuring the rate of release of mant-ADP from a protein, such as kinesin, that uses ATP for energy by breaking the high-energy phosphate bond. Mant-ADP is a fluorescent ADP (adenosine diphosphate) analog whose fluorescence is reduced when it is released from the enzyme. One can monitor the mant-ADP release directly by watching as the level of fluorescence decreases over time.
The HIV reverse transcriptase is a very fast enzyme. Thus, by using a combination of quench-flow and stop-flow methods, one can measure the individual rates of each step in the enzymatic pathway, look at the differences between strains of HIV, and then model each step in the enzymatic pathway. Understanding how the enzyme mutants treat the DNA substrates differently can help one model more precisely the interactions between molecules, their recognition modes, their chemistry, and ultimately, in the case of HIV, find a way to reduce or remove its threat to each of us.
Links
Kintek - The makers of stop-flow and quench-flow apparatuses.
Stop-Flow Kinetics in New Mexico
Molecular Kinetics Page
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