Creatine and Creatinine Metabolism - Physiological Reviews

1136 MARKUS WYSS AND RIMA KADDURAH-DAOUK Volume 80
IX. USE OF CREATINE ANALOGS AND
INVERTEBRATE PHOSPHAGENS AS TOOLS
FOR THE STUDY OF THE PHYSIOLOGICAL
FUNCTIONS OF THE CREATINE KINASE
SYSTEM
A. PCr in Comparison With Invertebrate
Phosphagens and Synthetic Analogs:
Thermodynamic and Kinetic Considerations
In the first account on PCr, Eggleton and Eggleton
(213) mentioned that “the supposed inorganic phosphate
of muscle is in certain conditions mainly organic phosphate
of a very labile nature [� PCr], which is so unstable
in acid solution.” This statement explains 1) why this
substance escaped detection before and 2) why it was
termed “phosphagen”. Whereas PCr was found predominantly
in vertebrates, a variety of other phosphorylated
guanidino compounds were subsequently identified in invertebrates
and were shown to play a physiological role
similar or identical to PCr in vertebrates (Fig. 8) (for
reviews, see Refs. 221, 226, 668, 669, 995, 1092, 1093).
Consequently, the term phosphagen nowadays is used
generally for all phosphorylated guanidino compounds
that may serve to regenerate ATP. PCr is unique in this
family, in so far as it is the only natural phosphagen with
a methyl group attached to the guanidino moiety of the
molecule (bold in Fig. 8). This feature may explain some
of the distinctive chemical properties of PCr.
All of the phosphagens are rather stable in alkaline
solution but are susceptible to acid hydrolysis, with the
acid lability decreasing in the order PCr � PArg � PTc �
PGc (see Refs. 226, 669). Although the rate of PCr hydrolysis
increases with increasing acidity, the rate of hydrolysis
of PArg, PGc, and PTc displays a maximum at pH
1.0–3.5. Acid molybdate accelerates the hydrolysis of PCr
but retards that of PArg, PGc, and PTc. Depending on
temperature, molybdate concentration, and pH, acid hydrolysis
of PCr results in the formation of Cr plus P i
and/or Crn plus P i. In contrast, no cyclic products are
formed upon acid hydrolysis of other phosphagens.
The N-methyl group of PCr eliminates almost all
resonance states in the guanidino group (221, 226). In
consequence, PCr is thermodynamically less stable (�G°�
for PCr hydrolysis ��44.6 kJ/mol at 35°C, pH 7.25 and 4
mM Mg 2� ) than PArg, PGc, PTc, and PL (�G°� ��39.4,
�41.4, �41.5, and �41.7 kJ/mol, respectively) (221). The
differences in thermodynamic stability between the various
phosphagens are also reflected in the different electronic
environments of the phosphorus nuclei as visualized
by 31 P-NMR. While the phosphorus nucleus of PCr
displays a chemical shift of �2.57 ppm relative to an
external standard of o-phosphoric acid, the respective
values for PArg, PGc, PTc, and PL are �3.00, �3.03,
�3.03, and �3.01 ppm, respectively (221).
The �G°� for PCr hydrolysis is also more negative
than that for ATP hydrolysis (�45.1 vs. �35.7 kJ/mol in
the absence of Mg 2� , �45.0 vs. �31.8 kJ/mol at 1 mM
Mg 2� ; pH 7.0, 38°C, I � 0.25 M) (538; see also Refs. 451,
474). This implies, under the assumption of near-equilibrium
conditions for the CK reaction in the cytosol, that
the free energy change (� affinity) of ATP hydrolysis
{defined as A � -dG/d� � ��G°� � RT ln([ATP]/
[ADP][P i]), where d� stands for the advancement of the
reaction} and thus the phosphorylation potential in a cell
can be buffered efficiently at much higher values by PCr
and Cr than by ATP and ADP alone. Buffering of the
phosphorylation potential, in turn, seems to be crucial for
some cellular processes, especially for the Ca 2� -ATPase
of the SR which depends on a free energy change for ATP
hydrolysis of at least 52 kJ/mol to allow proper muscle
relaxation (310, 451). The notion that CK may help in
maintaining high phosphorylation potentials in the intimate
vicinity of crucial ATPases is indeed supported by
the close functional coupling and the colocalization observed
for CK and Ca 2� -ATPase of the SR (see Ref. 646).
Based on the �G°� values for ATP and PCr hydrolysis,
the equilibrium constant for the reaction ATP � Cr 7
ADP � PCr, K� �([�ATP][�Cr])/([�ADP][�PCr]), where
� represents the sum of all ionized and Mg 2� -complexed
forms in solution and where pH is taken to be constant at
7.0, was calculated to be 37.8 in the absence of Mg 2� and
166 at a free Mg 2� concentration of 1 mM (38°C, I � 0.25
M) (538). Because K� critically depends on pH, temperature,
[Mg 2� ], and probably also on the concentrations of
other monovalent and divalent cations (see Refs. 287,
538), reported values for K� (e.g., Refs. 421, 518, 574)
should be taken as rough estimates and should not be
compared directly without considering differences in experimental
conditions.
It may be asked at this stage why evolution has
“chosen” PCr for the vertebrates and a variety of more
stable phosphagens for the invertebrates. Over many
years, the hypothesis that PArg is simply the evolutionary
precursor of PCr has attracted much attention (see, for
example, Refs. 1092, 1093). Evidently, this hypothesis implies
that PCr in some respect represents a functional
improvement over PArg. Even though the common ancestral
gene of all guanidino kinases may in fact have been an
arginine kinase (ArgK), only unsatisfactory arguments are
available for explaining the current distribution of the
various phosphagens in the animal kingdom.
PCr is the only phosphagen in vertebrates, but it is
also found in spermatozoa of a wide variety of invertebrates
(for reviews, see Refs. 221, 811, 1092). In these
invertebrate species, PCr in spermatozoa may coexist
with one or even two other phosphagens in other tissues
(pluriphosphagen phenomenon) (221, 668, 811, 1092). In
sea urchins of the class Echinoidea, for example, PCr is
the sole phosphagen in spermatozoa, whereas the differ-