Small-molecule allosteric activation of human glucokinase in the absence of glucose

Article abstract of DOI:10.1021/ml400061x

Synthetic allosteric activators of human glucokinase are receiving considerable attention as potential diabetes therapeutic agents. Although their mechanism of action is not fully understood, structural studies suggest that activator association requires

Full text of DOI:10.1021/ml400061x

Letter
pubs.acs.org/acsmedchemlett
Small-Molecule Allosteric Activation of Human Glucokinase in the
Absence of Glucose
Joseph M. Bowler,† Katherine L. Hervert,‡ Mark L. Kearley,† and Brian G. Miller*,†
†Department of Chemistry and Biochemistry, Florida State University, Tallahassee, Florida 32306, United States
‡Department of Chemistry, Ohio Wesleyan University, Delaware, Ohio 43015, United States
S
* Supporting Information
ABSTRACT: Synthetic allosteric activators of human gluco-
kinase are receiving considerable attention as potential
diabetes therapeutic agents. Although their mechanism of
action is not fully understood, structural studies suggest that
activator association requires prior formation of a binary
enzyme−glucose complex. Here, we demonstrate that three
previously described activators associate with glucokinase in a
glucose-independent fashion. Transient-state kinetic assays
reveal a lag in enzyme progress curves that is systematically
reduced when the enzyme is preincubated with activators.
Isothermal titration calorimetry demonstrates that activator
binding is enthalpically driven for all three compounds,
whereas the entropic changes accompanying activator binding can be favorable or unfavorable. Viscosity variation experiments
indicate that the kcat value of glucokinase is almost fully limited by product release, both in the presence and absence of activators,
suggesting that activators impact a step preceding product release. The observation of glucose-independent allosteric activation of
glucokinase has important implications for the refinement of future diabetes therapeutics and for the mechanism of kinetic
cooperativity of mammalian glucokinase.
KEYWORDS: Maturity onset diabetes of the young type II, glucokinase activators, kinetic cooperativity, hysteretic enzyme
uman glucokinase (GCK, hexokinase IV) catalyzes the
kinetic cooperativity of the enzyme. This GCK activator yielded
HATP-dependent phosphorylation of glucose in the first promising results in terms of therapeutic potential, enhancing
step of glycolysis.1 GCK is expressed in pancreatic β-cells where
both hepatic glucose metabolism and glucose-stimulated insulin
release from isolated rat islets. In vivo tests with diabetic rodent
models substantiated these results, showing reduced blood
glucose levels and augmented plasma insulin upon activator
treatment. To date, a variety of structurally unique allosteric
activators of GCK have been identified.8−11 One such molecule,
piragliatin (RO4389620), successfully advanced through phase
II clinical trials for the treatment of type 2 diabetes; however,
these trials were halted for undisclosed reasons.12,13
Structural studies indicate that GCK activators bind to an
allosteric site on the enzyme that is ∼20 Å removed from the
glucose binding site.14 Interestingly, the binding site for
allosteric activators overlaps with the location of many single-
site variants identified in patients with PHHI.15 Indeed, the
kinetic consequences of PHHI-associated mutations are
identical to those observed when the enzyme is assayed in
the presence of activator.4 The allosteric binding site is
noticeably absent in the crystal structure of unliganded GCK,
suggesting that small-molecule activation is dependent upon
glucose association. Consistent with this postulate, Ralph et al.
it functions as a sensor for glucose-dependent insulin
secretion.2 The unique kinetic features of GCK are directly
linked to its functional role in maintaining glucose homeostasis.
GCK displays mild positive cooperativity toward glucose and a
K0.5 value that approximates physiological glucose concen-
trations.3 The kinetic cooperativity of human GCK is
mechanistically interesting because the enzyme is a functional
monomer and contains only one binding site for glucose.4,5
The physiological importance of GCK is underscored by the
observation that mutations in the glk gene have a profound
impact on blood glucose levels. Inactivating glk mutations have
been directly linked to maturity onset diabetes of the young
type 2 (MODY-II), while mutations producing hyperactive
GCK variants cause persistent hyperinsulinemic hypoglycemia
of infancy (PHHI).6
As a result of its central role in glucose homeostasis, GCK
has emerged as an attractive target for diabetes therapeutic
development. In 2003, investigators from Hoffmann-La Roche
described the identification and in vivo characterization of a
lead compound for diabetes treatment that targeted GCK.7
Functional analysis revealed that this molecule operates as a
mixed-type allosteric activator, increasing the enzyme’s maximal
velocity, decreasing the K0.5 value for glucose, and reducing the
Received: February 13, 2013
Accepted: May 9, 2013
© XXXX American Chemical Society
A
dx.doi.org/10.1021/ml400061x | ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
failed to detect binding of activators to the unliganded enzyme
using differential scanning calorimetry and a scintillation
proximity assay.16 Moreover, a 2010 status update of GCK
activators suggested that these potential therapeutic agents
exclusively target the binary enzyme−glucose complex.12 In
contrast to these reports, one recent study supports the ability
of synthetic activators to associate with the unliganded
enzyme.17 To probe the mechanism of action of GCK
activators in more depth, we functionally characterized the
interaction of three previously described therapeutic lead agents
in the presence and absence of glucose.14,16−18 Our results
provide clear evidence supporting the ability of all three GCK
activators to function via a glucose-independent mechanism.
We synthesized GKA22 with an overall yield of 9% using a
six-step procedure adapted from a previous report (Figure S1,
Supporting Information).18 Details of the synthetic procedure,
as well as a description of the relevant intermediates, are
provided in Supporting Information. Two other synthetic
activators, Compound A and RO-28-1675, were commercially
obtained. To investigate the glucose dependency of activator
association, we conducted a series of experiments in which
activators were pre-equilibrated with the enzyme in the absence
of glucose. Previously, Neet and co-workers observed a lag in
enzyme progress curves when assays were conducted in the
presence of 30% glycerol. This lag was eliminated upon
preincubation with glucose, which was interpreted in terms of a
slow, glucose-induced conformational transition.19 We hy-
pothesized that if activators could associate with GCK in the
absence of glucose, a similar decrease in the lag would be
observed. To test this postulate, the activating properties of
each compound were evaluated using a standard assay for GCK
activity linking glucose 6-phosphate production to the
reduction of NADP+ via the action of glucose 6-phosphate
dehydrogenase. To exclude glycerol from assay mixtures, which
might promote activator association,11 we utilized a stopped-
flow apparatus to monitor early time points in the assay.
Reactions were initiated via the addition of 1 mM glucose and
monitored until the steady-state velocity was attained.
Consistent with previous reports, assay mixtures containing
only GCK produced progress curves showing a lag in the
attainment of steady-state; however, in the presence of
activators, we observed a reproducible decrease in the lag
(Figure 1). To quantify the lag for each condition, curves were
fitted to a single-exponential decay of one enzyme state to
another.19 Extrapolation of lifetime measurements from the fits
produced the transition time (τ), which reports on the extent of
the delay in reaching the steady-state regime. In the absence of
activator, the lag was characterized by a τ value of 0.51 min
(Table 1). When GCK was preincubated with GKA22 (20
Table 1. Thermodynamic and Kinetic Characterization of
GCK Activators at 25°C
μM), the τ value was reduced to 0.30 min. Compound A and
RO-28-1675 (20 μM) produced similar effects, yielding
transition times of 0.34 and 0.35 min, respectively. Notably,
at concentrations near the solubility limits of these compounds
(25−100 μM), we did not observe complete elimination of the
lag. It is unclear whether this failure is due to an inability to
saturate the enzyme with activator at these concentrations or
whether the activators are unable to fully shift the equilibrium
toward the more active species. Nevertheless, our results
demonstrate that activator association promotes an active
enzyme conformation, similar to the effects produced by
glucose. Importantly, this perturbation occurs without prior
glucose association, in the absence of glycerol. The ability of
activators to reduce the lag indicates that unliganded GCK
populates at least one conformational state capable of
associating with activators.
To fully characterize the enzyme−activator interaction, we
investigated the thermodynamics of activator association using
isothermal titration calorimetry (ITC). In the absence of
glucose, we did not detect activator binding at concentrations
approaching the solubility limits of these compounds. This
failure likely results from an inability to sufficiently populate the
GCK−activator complex at the activator concentrations (≤110
μM) and activator/enzyme ratios (≤10:1) possible in these
studies. By comparison, the activator/enzyme ratio used in the
transient-state assays described above equaled 2000:1. In the
presence of saturating glucose concentrations, association of
GKA22 and compound A with GCK is accompanied by a large,
favorable change in enthalpy and an unfavorable entropic
component (Figure 2, Table 1). Negative values for these
parameters indicate a binding event dominated by specific
interactions.20 In contrast, a previous study found that
interaction of the binary GCK−glucose complex with RO-28-
1675 and another synthetic activator involves favorable entropic
changes.21 Glucose binding to unliganded GCK is also
accompanied by a highly favorable entropic component (TΔS
= 10.7 kcal/mol).22 Structural studies provide no obvious
rationale for the differences in the entropic consequences of
activator association, although the differential extent to which
solvent is excluded from the allosteric binding pocket could
provide one explanation.23−29 Docking studies are consistent
Figure 1. Lag in the transient-state GCK progress curve obtained in
the absence of activator (filled circles) is reduced in the presence of
GKA-22 (open circles).
B
dx.doi.org/10.1021/ml400061x | ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
Figure 2. (A) Representative isotherm for GKA22 (110 μM) binding
to glucokinase (11 μM) in the presence of glucose. (B) Representative
isotherm for CpdA (100 μM) binding to glucokinase (10 μM) in the
presence of glucose. Thermodynamic parameters represent the average
of two or more independent experiments.
Figure 3. Normalized values of kcat as a function of relative solvent
viscosity in the absence (A) or presence of GKA-22 (B), RO-28-1675
(C), and CpdA (D). A slope of unity is consistent with product release
being fully rate-limiting.
with this possibility (Supporting Information). Despite differ-
ences in the thermodynamic details of activator association, the
ability of each activator to produce comparable reductions in
the lag indicates that activators elicit a similar kinetic effect.
All synthetic activators described to date have the ability to
as reflected by a slope of unity (Figure 3D). Together our data
suggest that activators such as RO-28-1675 and compound A,
which increase the maximal velocity of GCK, do so by
enhancing the rate of a step preceding product release.
Moreover, the magnitude of the slope of the normalized
value of kcat as a function of relative viscosity for unactivated
GCK suggests that the largest increase in Vmax that can be
expected from mechanistically similar activators is ∼30%. If
larger increases in the maximal velocity of GCK are a desirable
attribute of a diabetes therapeutic agent, an activator that
accelerates the rate of product release is needed.
The findings presented herein provide insight into the
mechanism of kinetic cooperativity in monomeric human
glucokinase. Current models suggest that GCK cooperativity
results from a slow, glucose-induced conformational reorgan-
ization of enzyme structure that takes place during the catalytic
cycle.32−34 One model for cooperativity, the ligand-induced
slow transition mechanism, posits a conformational selection
mode of ligand binding.32 Another cooperativity model, the
mnemonic mechanism, implies an induced fit mode of glucose
association.35 Although our present results do not allow us to
definitively distinguish between these two possibilities, the
ability of activators to associate with GCK in the absence of
glucose is consistent with the enzyme existing as a conforma-
tional ensemble whose population is shifted upon ligand
binding. In principle, any ligand or binding partner capable of
shifting the conformational equilibrium of unliganded GCK in
favor of the active state would function as an activator.
Our discovery that activators can function in a glucose-
independent fashion may also have implications for the
development of glucokinase-targeted therapeutic agents for
the treatment of type 2 diabetes. Clinical trials of one GCK
activator, piragliatin, were discontinued following phase II for
undisclosed reasons.12,13 One potential concern with this and
other activators is that these drugs might be too effective at
promoting glucose utilization, which could lead to hypoglyce-
mia.36 In light of our findings, it seems possible that long-term
administration of activators could lead to an enzyme that is
permanently primed for rapid glucose turnover. The physio-
logical consequences of such a condition are unknown,
especially in terms of GCK’s ability to associate with several
reduce the glucose K0.5 value. A subset of these compounds has
7−11
the added stimulatory attribute of increasing Vmax
.
Two of
the activators examined in this study, RO-28-1675 and
compound A, increase kcat by 30% and 10%, respectively.
GKA22 leaves kcat unaffected. As a first step in understanding
these differences, we used viscosity variation to probe the extent
to which the kcat value was limited by a diffusion-limited
process, such as product release, in the absence and presence of
activators.30 Using sucrose as a microviscogen, we conducted
GCK assays under saturating substrate conditions. A plot of the
normalized value of kcat as a function of relative solvent viscosity
in the absence of activator revealed that the kcat value is
fractionally dependent upon solvent viscosity, with a slope of
0.75 (Figure 3A). By comparison, we observed that the kcat
value of a truncated variant of GCK, for which chemistry is
expected to be fully rate-limiting,31 was independent of solvent
viscosity. These observations are consistent with the rate of
product release contributing substantially to the value of kcat for
unactivated GCK. In the presence of GKA22, which has no
measurable impact upon Vmax, the viscosity dependence of kcat
is unchanged (Figure 3B). Activation by RO-28-1675, which
produces the largest increase in Vmax, also results in no
measurable change in the dependence of kcat upon solvent
viscosity (Figure 3C). If the increase in Vmax caused by RO-28-
1675 results from an enhanced rate of product release, one
would expect the dependency upon solvent viscosity, as
reflected by the slope, to decrease. That activation by RO-28-
1675 does not alter the dependence of kcat upon relative
viscosity suggests that the increase in Vmax caused by this
compound involves acceleration of a step preceding product
release. Interestingly, compound A functions in a similar
manner, as it also fails to decrease the dependency of kcat upon
solvent viscosity. Instead, compound A appears to accelerate an
earlier step(s) in the catalytic cycle to such an extent that it no
longer contributes to the value of kcat. Thus, upon activation
with compound A, product release becomes fully rate-limiting,
C
dx.doi.org/10.1021/ml400061x | ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
F. Allosteric activators of glucokinase: potential role in diabetes
therapy. Science 2003, 301, 370−373.
(8) Sarabu, R.; Grimsby, J. Targeting glucokinase activation for the
treatment of type 2 diabetes: a status review. Curr. Opin. Drug
Discovery Dev. 2005, 8, 631−637.
(9) Johnson, D.; Shepherd, R. M.; Gill, D.; Gorman, T.; Smith, D.
M.; Dunne, M. J. Glucokinase activators: molecular tools for studying
the physiology of insulin-secreting cells. Biochem. Soc. Trans. 2007, 35,
1208−1210.
(10) Grimsby, J.; Berthel, S. J.; Sarabu, R. Glucokinase activators for
the potential treatment of type 2 diabetes. Curr. Top. Med. Chem.
2008, 8, 1524−1532.
(11) Matschinsky, F. M. Assessing the potential of glucokinase
activators in diabetes therapy. Nat. Rev. Drug Discovery 2009, 8, 399−
416.
(12) Matschinsky, F. M.; Zelent, B.; Doliba, N.; Li, C.; Vanderkooi, J.
M.; Naji, A.; Sarabu, R.; Grimsby, J. Glucokinase activators for diabetes
therapy. Diabetes Care. 2011, 34, S236−S243.
(13) Bonadonna, R. C.; Heise, T.; Arbet-Engels, C.; Kapitza, C.;
Avogaro, A.; Grimsby, J.; Zhi, J.; Grippo, J. F.; Balena, R. Piragliatin
(RO4389620), a novel glucokinase activator, lowers plasma glucose
both in the postabsorptive state and after a glucose challenge in
patients with type 2 diabetes mellitus: A mechanistic study. J. Clin.
Endocrinol. Metab. 2010, 95, 5028−5036.
(14) Kamata, K.; Mitsuya, M.; Nishimura, T.; Eiki, J.; Nagata, Y.
Structural basis for allosteric regulation of the monomeric allosteric
enzyme human glucokinase. Structure 2004, 12, 429−438.
(15) Pal, P.; Miller, B. G. Activating mutations in the human
glucokinase gene revealed by genetic selection. Biochemistry 2009, 48,
814−816.
(16) Ralph, E. C.; Thomson, J.; Almaden, J.; Sun, S. Glucose
modulation of glucokinase activation by small molecules. Biochemistry
2008, 47, 5028−5036.
(17) Antoine, M.; Boutin, J. A.; Ferry, G. Binding kinetics of glucose
and allosteric activators to human glucokinase reveal multiple
conformational states. Biochemistry 2009, 48, 5466−5482.
(18) McKerrecher, D.; Allen, J. V.; Bowker, S. S.; Boyd, S.; Caulkett,
P. W. R.; Currie, G. S.; Davies, C. D.; Fenwick, M. L.; Gaskin, H.;
Grange, E.; Hargreaves, R. B.; Hayter, B. R.; James, R.; Johnson, K. M.;
Johnstone, C.; Jones, C. D.; Lackie, S.; Rayner, J. W.; Walker, R. P.
Discovery, synthesis and biological evaluation of novel glucokinase
activators. Bioorg. Med. Chem. Lett. 2005, 15, 2103−2106.
(19) Neet, K. E.; Keenan, R. P.; Tippett, P. S. Observation of a
kinetic slow transition in monomeric glucokinase. Biochemistry 1990,
29, 770−777.
(20) Perozzo, R.; Folkers, G.; Scapozza, L. Thermodynamics of
protein−ligand interactions: history, presence, and future aspects. J.
Recept. Signal Transduction 2004, 24, 1−52.
(21) Anderka, O.; Boyken, J.; Aschenbach, U.; Batzer, A.;
Boscheinen, O.; Schmoll, D. Biophysical characterization of the
interaction between hepatic glucokinase and its regulatory protein. J.
Biol. Chem. 2008, 283, 31333−31340.
(22) Molnes, J.; Bjorkhaug, L.; Sovik, O.; Njolstad, P. R.; Flatmark, T.
Catalytic activation of human glucokinase by substrate binding−
residue contacts involved in the binding of D-glucose to the super-
open form and conformational transitions. FEBS 2008, 275, 2467−
2481.
(23) Takahasi, K.; Hashimoto, N.; Nakama, C.; Kamata, K.; Sasaki,
K.; Yoshimoto, R.; Ohyama, S.; Hosaka, H.; Maruki, H.; Nagata, Y.;
Eiki, J.; Nishimura, T. The design and optimization of a series of 2-
(pyridin-2-yl)-1H-benzimidazole compounds as allosteric glucokinase
activators. Bioorg. Med. Chem. 2009, 17, 7042−7051.
known binding partners including the glucokinase regulatory
protein and the pro-apoptotic protein BAD.11 The impact of
glucose-independent activation of GCK in the liver, another
prominent tissue where this enzyme is localized, is also unclear.
These uncertainties emphasize the need for continued
investigations into the mechanism of action of these important
putative therapeutic agents.
ASSOCIATED CONTENT
* Supporting Information
■
S
Synthetic procedures and experimental methods. This material
is available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*(B.G.M.) Phone: 850-645-6570. Fax: 850-644-8281. E-mail:
miller@chem.fsu.edu.
■
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Funding
This work was supported, in part, by a grant from the National
Institute of Diabetes and Digestive and Kidney Diseases
(DK081358).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We would like to thank Dr. Claudius Mundoma, Director of the
Physical Biochemistry Facility in the Institute of Molecular
Biophysics at Florida State, for help with ITC measurements
and Shehzad Khan-Thomas for synthetic assistance.
ABBREVIATIONS
■
GCK, glucokinase; MODY-II, maturity onset diabetes of the
young type 2; PHHI, persistent hyperinsulinemic hypoglycemia
of infancy; NADP+, nicotinamide adenine dinucleotide
phosphate; ITC, isothermal titration calorimetry
REFERENCES
■
(1) Wilson, J. E. Hexokinases. Rev. Physiol. Biochem. Pharmacol. 1995,
126, 65−198.
(2) Meglasson, M. D.; Matschinsky, F. M. New perspectives on
pancreatic islet glucokinase. Am. J. Physiol. 1984, 246, E1−E13.
(3) Holroyde, M. J.; Allen, M. B.; Storer, A. C.; Warsy, A. S.;
Chesher, J. M. E.; Trayer, I. P.; Cornish-Bowden, A.; Walker, D. G.
The purification in high yield and characterization of rat hepatic
glucokinase. Biochem. J. 1976, 153, 363−373.
(4) Cardenas, M.; Rabajille, E.; Niemeyer, H. Maintenance of the
monomeric structure of glucokinase under reacting conditions. Arch.
Biochem. Biophys. 1978, 190, 142−148.
(5) Larion, M.; Miller, B. G. Homotropic allosteric regulation in
monomeric mammalian glucokinase. Arch. Biochem. Biophys. 2011,
519, 103−111.
(6) Gloyn, A. Glucokinase (GCK) mutations in hyper- and
hypoglycemia: maturity-onset diabetes of the young, permanent
neonatal diabetes, and hyperinsulinemia of infancy. Hum. Mutat.
2003, 22, 353−362.
(24) Bebernitz, G.; Beaulieu, V.; Dale, B.; Deacon, R.; Duttaroy, A.;
Gao, J.; Grondine, M.; Gupta, R.; Kakmak, M.; Kavana, M.; Kirman,
L.; Liang, J.; Maniara, W.; Munshi, S.; Nadkarni, S.; Schuster, H.;
Stams, T.; St. Denny, I.; Taslimi, P.; Vash, B.; Caplan, S. Investigation
of functionally liver selective glucokinase activators for the treatment
of type 2 diabetes. J. Med. Chem. 2009, 52, 6142−6152.
(7) Grimsby, J.; Sarabu, R.; Corbett, W. L.; Haynes, N. E.; Bizzarro,
F. T.; Coffey, J. W.; Guertin, K. R.; Hilliard, D. W.; Kester, R. F.;
Mahaney, P. E.; Marcus, L.; Qi, L.; Spence, C. L.; Tengi, J.; Magnuson,
M. A.; Chu, C. A.; Dvorozniak, M. T.; Matschinsky, F. M.; Grippo, J.
D
dx.doi.org/10.1021/ml400061x | ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
(25) Nishimura, T.; Iino, T.; Mitsuya, M.; Bamba, M.; Watanabe, H.;
Tsukahara, D.; Kamata, K.; Sasaki, K.; Ohyama, S.; Hosaka, H.;
Futamura, M.; Nagata, Y.; Eiki, J. Identification of novel and potent 2-
amino benzamide derivatives as allosteric glucokinase activators.
Bioorg. Med. Chem. Lett. 2009, 19, 1357−1360.
(26) Mitsuya, M.; Kamata, K.; Bamba, M.; Watanabe, H.; Sasaki, Y.;
Sasaki, K.; Ohyama, S.; Hosaka, H.; Nagata, Y.; Eiki, J.; Nishimura, T.
Discovery of novel 3,6-disubstituted 2-pyridinecarboxamid derivatives
as GK activators. Bioorg. Med. Chem. Lett. 2009, 19, 2718−2721.
(27) Petit, P.; Antoine, M.; Ferry, G.; Boutin, J.; Lagarde, A.; Gluais,
L.; Vincentelli, R.; Vuillard, L. The active conformation of human
glucokinase is not altered by allosteric activators. Acta Crystallogr., Sect.
D: Biol. Crystallogr. 2011, 67, 929−935.
(28) Liu, S.; Ammirati, M.; Song, X.; Knafels, J.; Zhang, J.; Greasley,
S.; Pfefferkorn, J.; Qiu, X. Insights into mechanism of glucokinase
activation: observation of multiple distinct protein conformations. J.
Biol. Chem. 2012, 287, 13598−13610.
(29) Cheruvallath, Z.; Gwaltney, S.; Sabat, M.; Tang, M.; Feng, J.;
Wang, H.; Miura, J.; Guntupalli, P.; Jennings, A.; Hosfield, D.; Lee, B.;
Wu, Y. Design, synthesis and SAR of novel glucokinase activators.
Bioorg. Med. Chem. Lett. 2013, 23, 2166−2171.
(30) Brouwer, A. C.; Kirsch, J. F. Investigation of diffusion-limited
rates of chymotrypsin reactions by viscosity variation. Biochemistry
1982, 21, 1302−1307.
(31) Larion, M.; Miller, B. G. 23-residue C-terminal α-helix governs
cooperativity in monomeric human glucokinase. Biochemistry 2009, 48,
6157−6165.
(32) Frieden, C. Kinetic aspects of regulation of metabolic processes:
the hysteretic enzyme concept. J. Biol. Chem. 1970, 245, 5788−5799.
(33) Ainslie, G. R.; Shill, J. P.; Neet, K. E. Transients and
cooperativity. A slow transition model for relating transients and
cooperative kinetics of enzymes. J. Biol. Chem. 1972, 247, 7088−7096.
(34) Porter, C. M.; Miller, B. G. Cooperativity in monomeric
enzymes with single-ligand binding sites. Bioorg. Chem. 2012, 43, 44−
50.
(35) Ricard, J.; Buc, J.; Meunier, J. C. Enzyme memory: A transient
kinetic study of wheat-germ hexokinase. Eur. J. Biochem. 1977, 80,
581−592.
(36) Meiniger, G. E.; Scott, R.; Alba, M.; Shentu, Y.; Luo, E.; Amin,
H.; Davies, M. J.; Kaufman, K. D.; Goldstein, B. J. Effects of MK-0941,
a novel glucokinase activator, on glycemic control in insulin-treated
patients with type 2 diabetes. Diabetes Care 2011, 34, 2560−2566.
E
dx.doi.org/10.1021/ml400061x | ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX