Quorum Sensing

Determine the time it takes for the change inA 600 to level off.

This should be the minimum incubation time necessary to

reduce nonspecific, background reactions in the enzyme assay.

Record the progress curve for an additional 5 min, and calcu-

late the slope to determine background rate (Fig.2).

4. Repeat the experiment with acyl-ACP/acyl-CoA as the fixed
   substrate. Maintain acyl-substrate concentration between 100
   and 200μM and vary SAM over a broad range, between 10 and
   500 μM. After the change in absorbance levels off, continue to
   take measurements for an additional 5 min (Fig.2). The slope
   in the last 5 min of the progress curve is the background rate in
   ΔAbs/s.


5. Convert the slope toμM/min using the following relation (see
   Note 6):

ðÞrateΔAbs

s



Mcm

21,000



1

1cm



106 μM

M



60s

1min

 2 ¼ðÞrate

μM

min

6. The rate calculated instep 3must be the appropriate back-
   ground rate in the enzyme assay if SAM is maintained at a fixed
   concentration while the acyl-substrate concentration is varied.
   On the other hand, if acyl-substrate is the fixed substrate in the
   experiment, then the rate determined instep 4should be the
   background rate for the enzyme reaction (seeNote 6).

3.1.3 Determination
of Optimum Enzyme
Concentration

1. Set up the enzyme assay as referred to in Table1. Determine
   the initial rates for at least four different enzyme concentrations
   from 0.1 to 2μM.


2. The enzyme rate at the lowest acyl-ACP/acyl-CoA concentra-
   tion must be at least threefold higher than the background rate.
   The lowest substrate concentration in a substrate-velocity
   curve must be at least two- to threefold lower than theKmof
   substrate used in the experiment.


3. Choose the lowest enzyme concentration that meets the con-
   dition outlined instep 2.

3.1.4 Determination
of kcatand Km

1. Add 10buffer, 10SAM, nanopure water, DCPIP, and acyl-
   ACP/acyl-CoA to the cuvette (refer to Table1) and let the
   mixture incubate for the time determined in Subheading3.1.2.
   Add 10μl enzyme and collect the absorbance at 600 nm for
   5 min (Fig.2).


2. Calculate the linear slope between 0 and 200 s to determine the
   initial rate for each enzyme reaction. Please note that the data
   points in the first 30 s are usually excluded in the slope calcula-
   tion to account for sample mixing errors.

168 Daniel Shin and Rajesh Nagarajan