Name: P.ARJUN KUMAR NO: 10152, M.

E-1st year,

Molecular systems biology assignment 2

CHEM ENGG.

3) The Michaelis-Menten (MM) equation describes more or less how a large number of enzymes actually behave in real life. Should we expect anything different if we were to look at the behavior of a single enzyme molecule? Read the article: Ever-fluctuating single enzyme molecules: Michaelis-Menten equation revisited, Nature Chemical Biology 2006 Dec 25; 2: 87-94 a) What causes the non-single exponential nature of the waiting time distributions for the single enzymes and why does it only show up at high concentrations of the substrate. Ans) At higher substrate concentration we observe multiexponential behavior, and attributed this behavior of f(t) as dynamic disorder, which refers to fluctuations in the rate constants and is associated with the conformational changes due to substrate binding .At lower concentrations , enzyme substrate binding is rate limiting with pseudo-first order rate constant and hence f(t) in lower concentrations is monoexponential decay. however at higher substrate concentration becomes rate limiting and slow interconvertion among the conformers results in multiexponential decay of f(t). b) The authors argue that single enzymes undergo structural fluctuations over multiple timescales that leads to dynamic disorder? What does dynamic disorder imply and how do the authors treat dynamic disorder and so that MM kinetics still holds for single enzymes? Ans) Dynamic disorder mean that there are fluctuations in the rate constants and its associated conformational changes due to substrate binding.the authors premise is that interconverting conformers possess different enzymatic reactivities .therefore MM kinetics can be represented by a kinetic scheme involving confermers.In this scheme not only interconverts with and but does so with all other conformers as well. c) The single molecule MM equation is described by parameters 2 and CM. How does the definition of 2 account for variability in the values of k2. Why do they think that the disorder arises from distribution of values of k2 and not other rate constants? Ans) 2 =

( )

thus it represents the weighted harmonic mean of

for all

conformers, depending not only on the mean but also on the distribution of .it means that two mutations of an enzyme with an identical mean but different widths of the distributions would have the different 2.they think the disorder arises from distribution of values of k2 because as from the experimental plot it is quite obvious that at higher substrate concentrations it exhibits multiexponenetial decay. At higher concentrations enzyme substrate binding cannot become rate limiting, so whatever the deviation induced in the system from the expected monoexponential decay has to accounted in variation .and the second movement of f(t) is related to the randomness parameter (r).in the absence of dynamic disorder, r is unity at low or high substrate concentrations . and they had shown theoretically

that r rises above unity in the presence of dynamic disorder in k2,and at lower concentrations r is unity as expected. If we consider were in k-1 instead of k2, r would be larger than unity, which corroborates our original premise that dynamic disorder is present in k2.(dynamic disorder also cannot be present in k1 because since substrate concentration is held constant under experimental condition.) d) The authors describe a way to measure the slow fluctuations in the turnover rate constants of the single enzyme. What were the two main challenges of the measurement and how do the authors account for those? Ans) a.Single molecule enzymatic assays involving fluorescence detection have previously limited to short observations times because of photo bleaching of flourophores. To circumvent this problem, an assay with continuously replenished fluorescent product molecules was proposed. b. There is a strong background signal caused by resorufin (as these are produced by each turn over) molecules that are continuously generated by autohydrolysis even in the absence of the enzyme and that diffuse into the probe volume. In addition, resorufin molecules generated from previous or nearby enzymatic conversions can enter the probe volume. To circumvent this, they illuminated the area around the enzyme molecule with an intense laser beam to bleach resorufin molecules diffusing into the probe volume, thus suppressing the background signal atleast by two orders of magnitude. 2) Read the article: Direct observation of the rotation of F1-ATPase, Nature. 1997 Mar 20; 386(6622): 299-302F0-F1 ATP synthase is a Natures machine for making ATP. A portion of it called F1-ATPase can run in reverse direction, hydrolyzing ATP. This paper is the first demonstration that F1-ATPase is a rotary motor that runs uni-directionally and indefinitely. a) At low ATP levels, the angular motion of the motor is observed to be discrete (~ 120 deg). Combined with the shown structure of the protein complex, how many ATP molecules are expected to make one turn? Ans) The stoichiometry of three ATPase catalytic sites per single -subunit in the F1+ATPase indicates a rotary rate (with out load) of 1 revolutions per second at the cost of consuming 52 ATP per second this implies that it takes 3ATP for one complete revolution .but for the observed rate of filament rotation was at most 4 r.p.s and lower for longer filament by consuming the same amount of ATP molecules from this we can conclude that no of ATP molecules required for 1 complete revolution is around 52 4 13 ATP molecules(under load). b) Why does the F1-ATPase show Brownian motion in absence of ATP but only unidirectional motion in presence of the ATP? By comparing the discussion on Kinesin in the class, describe the rotatory motion of the F1-ATPase. Ans) The ATP synthase operates through a mechanism in which the three active sites undergo a change in binding affinity for the reactants of the ATP-ase reaction, ATP, ADP and phosphate. The change in affinity accompanies a change in the position of the gamma-subunit relative to

the a, b-ring, which involves a rotation of the one relative to the other. In the direction of ATP synthesis, the rotation is driven by a flux of H+ down the proton gradient, through a coupling between the g-subunit, and the c-subunit of FO.but in the case of Kinesin the structural asymmetry also contribute to the forward motion along the filament.

c) The torque generated by the motor can be estimated by using actin filaments of various lengths. Describe how this was done to calculate the force exerted by the motor and how the drag force is related to the length of actin filament. Ans) We can calculate force generated by dividing the torque with the radius. the expression for torque is given in paper for the propeller rotation as

Drag force is defined as the force exerted by the fluid on the surface of the solid in direction of flow, so as the length of the filament is increased drag force exerted also increases and vice versa.,(it means that at higher drag for the longer filament, it is rotated against a heavy load produced by the hydro dynamic friction ) d) Can you use the information presented in this paper to argue that the F1-ATPase is a rotary motor with essentially 100 % conversion efficiency of chemical energy into mechanical work? Ans) Using the expression of the torque we can calculate the amount of workdone required for 120 deg step moving the actin filament under load(hydrodynamic friction) or the work per ATP molecule. And this should be compared with the free energy of the ATP hydrolysis and then we can comment on the above question. in the given paper author didnt mention anything about the energy release during the ATP hydrolysis, but from the data it is around 90 pN nm per ATP molecule and the work done in a 120 degree step, or the work per ATP molecule, is thus ca. 80 pN nm so these figures corroborates that F1motor can act as 100% efficient motor(fully reversible in nature).