On Fri, 1 May 1998, Petr Kuzmic wrote:
> claude aflalo wrote:
>> > May I take the liberty of mentioning the following publications of
> > mine which may be relevant to the issue of the analysis of function for
> > localized enzymes:
> >
> > C. AFLALO & N. SHAVIT, 1982. Source of Rapidly Labeled ATP
> [snip]
> > C. AFLALO, 1997. Localized firefly luciferase probes ATP
>> Michael Kohn's original question was "How are reaction kinetics affected
> by binding to a catalyst (...) to a surface?". I wonder if there is any
> possibility that you could kindly distill a concise answer (an
> "abstract") from your numerous publications on luciferase. If so, I
> myself would be very greatful.
>> The particular question I am interested in has to do with enzyme
> -inhibition-, e.g., by pharmaceutical agents. How relevant are studies
> of in-vitro inhibition activity (measurements of inhibition constants in
> the solution phase) when the targed enzyme in immobilized? Any example
> data?
>> --Petr
>OK, I'll try to keep concise...
The rationale behind cellular enzymology is that enzymes there are mostly
at interfaces between cellular surfaces (membranes, cytoskeletal fibers,
multienzyme complexes) and a limited aqueous phase. This applies also to
so-called soluble (e.g. cytosolic) enzymes.
Thus the rules of classical enzyme kinetics do not necessarily apply in
situ, and the kinetic behavior of intracellular enzymes and metabolic
pathways must also be influenced (to variable extent) by physical steps
or phenomena (diffusion, partition of both catalysts and reactants) along
with the rules of heterogeneous catalysis.
Considering the minimal system of an enzyme immobilized at a surface, for
efficiently transformed substrates (high catalytic efficiency kcat/Km), it
should be expected that the substates would be depleted, and the products
would accumulate in the vicinity of the membrane just because of
diffusional resistance, relative to their respective concentrations in the
bulk solution.
The latter, measured by standard methodologies, are normally used to
derive kinetic properties of the immobilized enzyme according to
homogeneous catalysis rules, which are based on the tacite assumption that
diffusion and other physical limitations are negligible.
The EFFECTIVE kinetic behavior of an enzyme is ultimately governed not
only by INTRINSIC kinetic parameters (kcat, Km under optimal conditions),
but also by reactant concentrations and other environmental factors (pH,
ionic strength, etc.) as they are "sensed" by the enzyme ("local"), which
may be different than those occurring in solution ("bulk", determined by
the experimenter), due to the physical state of the enzyme in its
INHERENT environment (e.g. immobilized vs. in dilute well-mixed solution).
Thus, the behavior of an immobilized enzyme may appear quite different to
an external observer as compared to a local one, on the one hand, and to
that observed in solution, on the other hand. As a corollary, the
behavior of an immobilized enzyme observed locally should ideally be
identical to that in solution.
I used firefly luciferase as a probe for monitoring steady state [ATP] in
a cyclic heterogeneous coupled system. ATP is synthesized by oxidative
phosphorylation in yeast mitochondria (surface source) and consumed in
solution by hexokinase (bulk sink). ATP may be monitored either in
solution (soluble luciferase) or at the surface of mitochondria
(genetically engineered localized luciferase). 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).
The extent and direction of the departure in kinetic behavior of an
immobilized enzyme from that observed with the enzyme in solution would
depend on inherent properties of the support. 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).
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.
This whole "lecture" has gone quite off control, but I hope that the
rationale remains clear. I hope too that this won't initiate World War
III...
Sincerely,
Claude
Claude Aflalo ######################## Phones: Office 972-7-6472118 ####
Dept. of Life Sciences Lab 972-7-6472119
Ben Gurion University of the Negev Fax 972-7-6472890
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