Introduction to Enzyme and Coenzyme Chemistry - E-Library Home

60 Chapter 4
E + S
K i
EI+ I
ES
E + P
k i
E-I
Act
1/v i
slope K i /k i
v i
1/k i
time
1/[I]
Figure 4.7 Irreversible inhibition.
ENZ
observation cuvette
rapid mixing device
ENZ + S
S
stopping syringe
observe time-course of enzymatic reaction
by UV or fluorescence spectroscopy
Figure 4.8 Stopped Xow apparatus.
This apparatus consists of two syringes containing: (i) substrate and (ii) a
stoichiometric amount (i.e. 1–100 nmol) of enzyme, connected to a rapid mixing
device. The syringes are driven so as to Wll the observation cuvette with freshly
mixed enzyme and substrate. At this point, the reaction time of the mixture is
given by the distance from the mixer and the linear Xow rate. The syringes are
physically stopped, and the enzyme/substrate mixture is allowed to react along
the enzymatic reaction pathway. Changes in absorbance or Xuorescence in the
observation cuvette are measured on a 10–1000-ms timescale, giving a complete
time course of a single catalytic cycle of the enzymatic reaction. Several substrate
concentrations are used to enable individual rate constants to be measured. In
this way a multi-step enzymatic reaction can in principle be broken down into its
individual steps, the slowest of which (the rate-determining step) should correspond
to the steady state k cat value measured by steady state kinetics.
4.4 The stereochemical course of an enzymatic reaction
The vast majority of biological molecules are chiral, that is a molecule whose
mirror image is non-superimposable upon the original. In the case of carbonbased
compounds, if a carbon atom is surrounded by four diVerent groups,