Petr Kuzmic wrote:
>Discussions about the mythical "Rate Limiting Step" in traditional
>(single-enzyme) enzymology seem to take place repeatedly, I suppose ever
>since Haldane published his "steady-state approximation" of enzyme
>catalysis in 1926. The issue is whether a particular step in a
>catalytic mechanism for the given enzyme (as opposed to a particular
>enzyme reaction in a multienzyme regulation cascade) can be declared as
>somehow "rate limiting". A great deal of confusion is seen on this
>issue, similar to the RLS in MCA.
>MCA can be relatively simply be adapted to analyse rate-limitation by steps
or rate constants within single enzymes (or any other molecular process
such as transporter or chemical reactions). One simply defines the extent
of rate limitation by a step or rate constant over the enzyme rate as
usual for flux control coefficients (approximately the % change in
enzyme rate for a 1% change in the steps rate constants). This
coefficient quantifies the extent of rate limitation and obeys all kinds
of nice theorms e.g. the coefficients of all steps add up to one, and
are related to the thermodynamics of steps, the relative size of rate
constants, and to intermediate levels. These relations then enable one
to EXPLAIN why certain steps are limiting or not. They also show that
previous criteria used to identify rate-limiting steps (such as slowest
step) are missleading or plain wrong. If all the rate constants are
known all the control coefficients can be simply calculated.
We did this for a number of enzymes whose rate constants have been
reported in the literature, and found (unsuprisingly) that control
was shared between a number of steps, and this extent of control
varies with condition (such as substrate and product concentrations).
More surprisingly the rate-limiting step identified by the authors of
the papers supplying the rate constants were usually identified wrongly,
i.e. it is quite difficult to "see" were rate limitation is even if
you have all the rate constants if you do not actually calculate them
- rate limitation is a system property.
There was no classical rate-limiting step in any of the enzymes we
analysed - which led us to conclude that the concept of a rate-limiting
step is useless. However, I am still worried that our sample of
enzymes was biased, as we used only those where all rate constants
had been measured. Possibly no one would bother or try to measure
rate constants for an enzymes that trully had a rate-limiting step.
It would be useful to try and make a more systematic survey.
We have not got arround to applying the analysis experimentally
which would be nice. This would require an enzyme where the significant
intermediates could be measured relatively easily in the steady state.
Any ideas?
Dr Guy C. Brown
Department of Biochemistry,
University of Cambridge,
Tennis Court Road,
Cambridge CB2 1QW, UK.
Tel: (44) (0)1223 333342
Fax: (44) (0)1223 333345
e-mail: gcb at mole.bio.cam.ac.ukhttp://www.bio.cam.ac.uk/dept/biochem/brown
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