claude aflalo wrote:
>> On Fri, 1 May 1998, Petr Kuzmic wrote:
>> > [...] How relevant are studies
> > of in-vitro inhibition activity (measurements of inhibition constants in
> > the solution phase) when the target enzyme is immobilized?
> As a corollary, the
> behavior of an immobilized enzyme observed locally should ideally be
> identical to that in solution.
This probably implies a physical picture in which the immobilized enzyme is
floating around attached to a -tether-. If I simply snip the tether, and then
study a soluble form of the previously immobilized enzyme [as is often done],
I should observe very little change in catalytic properties. However, if an
enzyme is embedded in a multienzyme complex or partially burried in the
membrane itself, I suppose that this must change the structure (tertiary, or
even secondary) of the protein molecule. I supposed that under those
circumstance the behavior of the solubilized and immobilized enzyme should be
intrinsically different...
> Experimental measurements
> indicate that by increasing hexokinase concentration added to the medium,
> while bulk [ATP] detected by soluble luciferase becomes vanishingly small,
> its local concentration (detected by localized luciferase) tends to a
> non-zero, finite value which is not decreased further by additional
> hexokinase. The results are consistent with a limiting mass transfer of
> nucleotides (ATP and ADP) between bulk solution and the outer membrane of
> mitochondria (upon which ATP appears and accumulates, while ADP disappears
> and is depleted, relatively to bulk solution).
I think that is what Gary Nelsestuen is saying in
@article { Nels9781
author = "G. L. Nelsestuen and M. B. Martinez",
title = "Steady State Enzyme Velocities That Are Independent
of [Enzyme]: An Important Behavior in Many Membrane
and Particle-Bound States",
journal = "Biochemistry",
volume = "36", pages = "9081-9086", year = "1997" }
It makes perfect sense to me. I think that directly at the solid/liquid
interface, the substrate and product molecules will be frozen in place by
adsorption, so not even diffusion [much less than laminar flow, which is zero
directly at the interface] can move them.
> For instance, fixed negative
> charges on the support should yield increased or decreased apparent Km's
> for negatively or positively charged substrates, respectively, due to
> their electrostatic partition (equilibrium) between the microenvironment
> of the enzyme and the bulk solution, independently of - and in addition to
> - diffusional resistance (steady state).
[I suppose that is just one example of how what I called 'adsorption' might happen.]
> The interpretation of macroscopic (bulk) measurements of a localized
> enzyme is difficult since the properties of the support (say a biological
> membrane) are often poorly defined. It is however sensible that a change
> in an apparent kinetic constant (say Ki) may be attributable to INHERENT
> properties of the enzyme localized on a membrane.
Claude, have you (or anyone you are aware of) ever measured inhibition
constants for an immbilized enzyme, then took the same or equivalent enzyme
and repeated the study in solution?
-- Petr
_____________________________________________________________
Petr Kuzmic Ph.D. * BioKin Ltd. * Madison, WI 53708-8336, USA
pkuzmic at biokin.com * http://www.biokin.com * 608.256.1269 fax
