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418 Opinion TRENDS in Biochemical Sciences Vol.26 No.7 July 2001
Table 1. Catalyzed reactions involving substrate-like and product-like states of filament assembly mediated by profilin is
differing in their noncovalent bonding and/or position sufficient to maintain the fast rates of actin-based
Reaction type Examples cell motility, and any greater rate enhancement
offers no selective advantage.
Facilitated exchange of Profilin-promoted exchange of actin-
protein-bound ligands bound nucleotides
A more encompassing definition of enzyme catalysis
Exchange factors acting on G proteins
As there are so many instances in which biological
Chaperonin-mediated reactions Protein folding
catalysis is not attended by changes in covalent
Assembly of multi-subunit protein complexes
bonding, I offer a broader definition: enzymes
Molecular motors Myosin locomotion on actin filaments
catalyze the making and/or breaking of chemical
Kinesin and dynein locomotion on microtubules
bonds by promoting substrate and/or product access
Cytoskeleton self-assembly ATP-dependent actin filament assembly
to the transition state. (This statement deliberately
GTP-dependent microtubule self-assembly
avoids specifying how an enzyme promotes catalysis;
Polymerase processivity ATP-dependent clamp-loading onto DNA for example, by stabilizing enzyme transition states,
polymerases
destabilizing the ground state, reorganizing active-
Contact-transfer polymerization of actin during
actin-based motility
site solvent molecules, enabling fluctuating motions
within protein domains, vibrationally coupling
Active transport Sodium and/or potassium ATPase
atomic motions, and managing entropic and/or
ATP synthase
enthalpic contributions.)
Carrier-mediated transport Sugar transport
Although this definition seems no more
Amino acid transport
encompassing than existing definitions of enzyme
catalysis, the crucial difference is the use of the
Motile cells have developed a motility complex phrase ‘chemical bond’ in place of ‘covalent bond’.
that uses profilin to mobilize actin–ATP in the form In his book The Nature of The Chemical Bond10,
of profilin–actin–ATP, thereby accelerating the actin Linus Pauling offered the following guiding
polymerization rate by a factor of 200–500. Again, comment: ‘We shall say that there is a chemical
profilin acts catalytically without any effect on the bond between two atoms or groups of atoms in case
equilibrium constant for actin filament assembly8. that the forces acting between them are such as
Actin-based cell crawling has a limiting rate of to lead to the formation of an aggregate with
1 µm s−1, corresponding to the addition of ~500 sufficient stability to make it convenient for the
actin monomers per second to the growing end of chemist to consider it as an independent molecular
each actin filament9. Based on the intracellular species.’ Because many protein conformational
concentration of actin monomers, the enhancement states and numerous protein–ligand complexes are
sufficiently long-lived to exhibit chemically
(a) X‡ (b) definable properties, their formation and/or
transformation must be considered as chemical
Uncatalyzed reactions. Thus, by admitting a more encompassing
definition of enzyme catalysis, it becomes clear that
profilin is a remarkably versatile enzyme, one that
speeds up two entirely different reaction types
attended by changes in noncovalent-bonding
Gibbs free energy
interactions.
There are other special cases of enzyme catalysis,
Uncatalyzed including those agents that catalyze noncovalent
EX‡
P‡ self-assembly of macromolecular and
Catalyzed F–P‡
supramolecular structures. In this context, catalysts
of protein folding and refolding, cytoskeletal filament
Catalyzed assembly and chromatin condensation should be
E+S E–P F–PT + D regarded as enzymes. With modest tinkering,
F + PD + T F–PD + T F + PT + D
E–S Pauling’s prescient comment can be extended to
E+P
include the persistent, chemically definable position
Progress of reaction
Ti BS
of a solute relative to the faces of a membrane. In this
way, the proposed definition of enzyme catalysis also
Fig. 1. Typical reaction coordinate diagrams for catalyzed and uncatalyzed reactions involving changes treats membrane transporters as specialized
in covalent or noncovalent bonding. Except for differences in the magnitude of the activation energy
(∆Eact), reactions involving changes in covalent and/or noncovalent bonding follow the same basic
enzymes. The Enzyme Commission now treats
reaction scheme. (a) Classical enzymatic process showing progress of the classical covalent bond- transporters as enzymes, recognizing that a change
altering reaction in the absence and presence of enzyme catalysis. (b) Facilitated ligand exchange, in the position of a metabolite with respect to a
showing progress of the ligand-exchange reaction. Abbreviations: D, nucleoside 5′-diphosphate;
membrane defines substrate-like and product-like
E, enzyme; F, exchange-promoting factor; P, nucleotide-binding protein; PD, protein–nucleoside
5′-diphosphate complex, P‡, protein with vacant nucleotide site; PT, protein–nucleoside states. Boyer’s binding change mechanism for ATP
5′-triphosphate complex; S, substrate; T, nucleoside 5′-triphosphate; X, reaction intermediate. synthase11 and the Mackinose–Jencks calcium pump
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Opinion TRENDS in Biochemical Sciences Vol.26 No.7 July 2001 419
model12,13 certainly reinforce the idea that stoichiometric coupling of tubulin incorporation and
transporter mechanisms are inherently similar to GTP hydrolysis occurs under normal physiological
other enzyme mechanisms. conditions, but is lost in the presence of colchicine,
which blocks microtubule self-assembly14,15. Tubulin
Energases: a distinct class of enzyme-catalyzed reactions only exhibits an enhanced capacity to hydrolyze GTP
Discoveries of the past two decades have convincingly in the presence of colchicine. Similar statements
demonstrated the pervasiveness of mechanochemical apply to the abilities of other uncoupling agents to
proteins that transduce the Gibbs free energy of increase the hydrolase activities of ATP synthase and
nucleotide hydrolysis into some form of useful work. other ATP-dependent transporters.
The product of these reactions can be described as a Another relevant example of energase action is the
form of translational movement, rotation or solute ATP-dependent clamp-loader that permits DNA
gradient. Under normal physiological conditions, polymerases to remain associated with the DNA
nucleotide hydrolysis is stoichiometrically coupled to template through many rounds of phosphodiester
the production of an increment of useful work. bond synthesis. Upon loading the lock-washer-shaped
‘Energase’ is offered as a new term that: (1) treats clamp, DNA polymerase cannot dissociate from its
these mechanochemical systems as a distinct enzyme DNA template, and the polymerase processively
class; (2) uses a root word (energy) known in all replicates several thousand bases before
modern languages; and (3) reinforces the idea that dissociating16–18. As the holoenzyme moves along the
the energy of changes in chemical bonding is replication fork, the polymerase continuously extends
transduced into mechanical energy. The term DNA on the leading strand; however, a second
energase also fits nicely alongside oxidase, reductase, polymerase molecule acting on the lagging strand
hydrolase, lyase, isomerase and ligase. releases its sliding clamp upon synthesis of each
The present Enzyme Commission classification Okazaki fragment. The polymerase must then return
does not explicitly account for the noncovalent ‘work to the advancing replication fork where it reattaches
steps’, thereby treating energases as hydrolases. That to another clamp that the clamp-loader has deployed
energases constitute a separate class becomes at an upstream RNA primer. The clamp-loader does
obvious when we consider equilibrium constants for not merely hydrolyze ATP, it is an energase that
three different ATP-dependent reactions (Table 2), accomplishes an increment of mechanical work as it
without specifying how metal ions are involved or how attaches the clamp around DNA.
protons are released. The fundamental nature of the The GTP-regulatory protein superfamily of
mechanical work step in energase reactions is GTPases19,20 represents yet another example of
illustrated by treating conformation-state1 as a energases that use the Gibbs free energy of GTP
substrate-like species and conformation-state2 as a hydrolysis to modulate the affinity of noncovalent-
product-like species. The relative abundance of these binding interactions. Effective G-protein-mediated
conformational states {i.e. [state2]/[state1]} must be regulation of hormone receptors stems from GTPase-
explicitly indicated when writing the chemical linked modulation of agonist and antagonist potency.
reaction and the equilibrium constant. Although the G proteins are enzymes that exist in at least two
quotient [ADP][Pi]/[ATP] is common to all three states (Eqn 2):
reactions, energases actually share much more in
common with synthases than with hydrolases. One [Interaction state1] G–GTP → [2]
simply cannot ignore the substrate-like and product- [Interaction state2] G–GDP + Pi
like species that differ only in the energetics of their
noncovalent interactions. in which the strength of noncovalent interactions
Ironically, the hydrolytic activities of energases depends on the energy difference between the
often become exaggerated when steps in an energase interaction sites.
reaction are uncoupled by various treatments or A characteristic feature of all catalyzed processes
agents. A good example is the tubulin GTPase. Strict is that the unmodified catalyst is regenerated after
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420 Opinion TRENDS in Biochemical Sciences Vol.26 No.7 July 2001
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Opinion TRENDS in Biochemical Sciences Vol.26 No.7 July 2001 421
8 Kang, F. et al. (1999) Profilin promotes barbed-end 14 MacNeal, R.K. and Purich, D.L. (1978) 19 Gilman, A.G. (1987) G proteins: transducers of
actin filament assembly without lowering the Stoichiometry and role of GTP hydrolysis in receptor-generated signals. Annu. Rev. Biochem.
critical concentration. J. Biol. Chem. 274, bovine microtubule assembly. J. Biol. Chem. 56, 615–649
36963–36972 253, 4683–4687 20 Vale, R.D. (1996) Switches, latches, and
9 Stossel, T.P. (1993) On the crawling of animal 15 Purich, D.L. and Angelastro, J.M. (1994) amplifiers: common themes of G proteins and
cells. Science 260, 1086–1094 Microtubule dynamics: bioenergetics and control. molecular motors. J. Cell Biol. 135, 291–302
10 Pauling, L. (1945) Resonance and the chemical Adv. Enzymol. 69, 121–154 21 Leyh, T.S. (1999) On the advantages of imperfect
bond. In The Nature of the Chemical Bond, p. 3, 16 Kelman, Z. and O’Donnell, M. (1995) DNA energetic linkage. Methods Enzymol. 308, 48–70
Cornell University Press polymerase III holoenzyme: structure and 22 Grubmeyer, C.T. et al. (1999) Energy coupling
11 Boyer, P.D. (1997) The ATP synthase – a splendid function of a chromosomal replicating machine. through molecular discrimination: nicotinate
molecular machine. Annu. Rev. Biochem. 66, Annu. Rev. Biochem. 64, 171–200 phosphoribosyltransferase. Methods Enzymol.
717–749 17 Kornberg, A. and Baker, T. (1992) Prokaryotic 308, 28–48
12 Makinose, M. (1973) Possible functional states of DNA polymerases other than E. coli Pol I. In DNA 23 von Hippel, P.H. and Delagoutte, E. (2001) A
the enzyme of the sarcoplasmic calcium pump. Replication, 2nd edn, p. 165, W.H. Freeman general model for nucleic acid helicases and their
FEBS Lett. 37, 140–143 18 Bertram, J.G. et al. (2000) Molecular mechanism ‘coupling’ within molecular machines. Cell 104,
13 Jencks, W.P. (1989) How does a calcium pump and energetics of clamp assembly in Escherichia 177–190
pump calcium? J. Biol. Chem. 264, coli. The role of ATP hydrolysis when γ complex 24 Koshland, D.E., Jr (1960) The active site and
18855–18858 loads β onto DNA. J. Biol. Chem. 275, 28413–28420 enzyme action. Adv. Enzymol. 22, 45–98
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