As catalysts of chemical transformation, enzymes convert large amounts of
substrate to product without undergoing permanent alteration themselves.
Changes that do occur must be readily reversible so that the enzyme can be
recycled for further use. Such transient changes may be conformational or
chemical and are rarely detected, perhaps because they are too rapid. However,
they are of great interest since they reflect changes that occur in the
catalytic process. Whenever recycling occurs, it may be the event that is
influenced by regulatory factors. In a well established example, carbonic
anhydrase requires the hydroxyl group of Zn-OH to be protonated from solvent
in order to regenerate the Zn form of the enzyme for reaction with
bicarbonate. Other examples are known in which recycling is too rapid to study
well. An exception to this is metal-independent fumarase for which recycling
is rate-limiting at even low turnover rates.
The initial idealized model
for the fumarase reaction is shown in Scheme 1, where M=malate,
F=fumarate, Em=malate-specific enzyme, and Ef=fumarate-specific enzyme.
The dehydration is shown as a single rapid interconversion, step 1,
with recycling between Ef and Em. Further progress has been made in
characterizing step 2 and explaining the activation that occurs with
either substrate at high concentration. The rate of recycling can be
determined by the substrate-induced countertransport method (counterflow)
in which a solution of tracer M* and F* in equilibrium is added to enzyme
with unlabeled substrate, either M or F. For example, as substrate M is
converted to F, the labeled product F* counterflows if the steady state
ratio Ef/Em remains above its equilibrium due to slow recycling.
The extent of displacement of the M*/F* equilibrium gives the rate constant
for the slow direction of recycling, k2 or k-2.
Using D2O instead of H2O,
recycling was slowed two-fold, opposite to the direction found
with a viscosity control. The recycling rate was activated by
the acidic component of buffers and by low pH suggesting that H+
transfer from the solvent to the enzyme occurs in the slow step
of recycling. A reasonable minimum mechanism would require two
proton transfers, Scheme 2. An acidic hydrogen will be required
to abstract the C2-OH of malate which is known to be lost as H2O
before fumarate is released. Therefore, an H+ must be recovered
in each cycle to form Em. The C3-H of malate is retained by Ef,
as shown previously by intermolecular 3H transfer. This H+ must
be lost in each cycle, step 2, in order to generate the base
required for the next malate dehydration.
Evidence for the recycling intermediate, E, is as follows: A competitive
inhibitor, mesotartrate increases the D2O effect measured in the
M to F direction but decreases it when measured in the hydration of fumarate.
This is expected if mesotartrate binds most tightly to the non-specific
intermediate E. In addition, counterflow, shown with M* alone, was greater
in D2O implying an increase in EH in the hydration of F. Inhibitors that
bind to E and allow it to accept a proton: E-I + H+ going to
EH-I going to I + EH will have the effect of decreasing counterflow, since while
this recycling is occurring there is no competitive counterflow possible due
to the bound inhibitor.
Interaction of malate or
fumarate with E provides an explanation for activation by
high substrate, previously attributed to cooperativity
or allosteric activation. These new complexes must be
functional, since neither substrate is known to form an
inhibitory complex. As shown in Scheme 3, these new steps
are equivalent to assuming that the complexes
EH-M and EH-F undergo random dissociation of proton
or substrate. This property has already been noted in
tritium-transfer experiments in which less than complete
intermolecular transfer could be accomplished especially in
the presence of buffer bases. These alternative pathways explain the
biphasic character of the equilibrium exchange rate when measured as a
function of concentration of M + F. Negative cooperativity occurs
because the slow steps of recycling are bypassed at high
concentration of substrate. This explanation replaces the
need for unique activating sites for which evidence has been
lacking with either the pig heart or Escherichia coli
enzymes. It is likely that the D2O effects that are observed
at high substrate concentration and in equilibrium exchange
are largely the result of reprotonation of enzyme ligand complexes,
most likely 
. Observations have been made
attempting to explain the activation of fumarase by monoanions
such as Cl- and acetate-. These ions are much more effective
with fumarate than malate; they increase recycling and eliminate
the phenomenon of activation by substrate giving
standard Michaelis kinetics. Our explanation for the effect
of inhibitors on counterflow may pertain here as well. By
combining with E, these anions facilitate proton transfer so
that recycling becomes rapid. Monoanions are not
inhibitors at concentrations that stimulate recycling,
which may mean that recycling can occur rapidly during a
very brief occupancy of the enzyme. The dianion orthophosphate
binds more tightly and is a competitive inhibitor
as well as an activator.
A major goal of our work is to
determine how protons are used in enzymatic reactions and how they are made
available from the solvent for each reaction cycle. In most cases, the
chemistry is slow and recycling is too fast to study. The fumarase enzymes
provide an example of the reverse case, offering an opportunity for the
detailed study of how protons travel between solvent and active sites, a
fundamental step in many enzyme reactions. The recycling mechanism of fumarase
that has been elaborated helps to explain the mode of action of several
activators and inhibitors.