F. Glufamine Synthetase
VII. ALLOSTERIC INHIBITION IN ENZYME COMPLEXES AND MEMBRANE SYSTEMS
A case of a complex of two enzymes exhibiting control by a common negative effector (UTP) has been mentioned (135). Yeast aspartate transcarbamylase and carbamyl phosphate synthetase were copurified together with a common regulatory protein. These three units are appar
ently controlled by a single gene. This complex appears to represent a physiological association of two adjacent enzymes of the pyrimidine biosynthetic pathway. The inhibitor UTP stabilized the regulatory site of the aggregate since the sensitivity of the aspartate transcarbamylase moiety to regulation was diminished during purificaton in the absence of this effector. This desensitization process was accompanied by dissocia
tion of the intact complex (MW about 800,000) into a half-complex (MW 380,000) lacking aspartate transcarbamylase sensitivity to UTP during chromatography on a gel filtration agent (135).
A complex containing phosphorylase a and glutamic-pyruvate trans
aminase activity has been purified and studied (141)- In this aggregate, an activator of phosphorylase a, AMP, was able to transmit its effect to the transaminase at 37°C. Glucose, glucose 1-phosphate, and glycogen
inhibited the transaminase activity but the inhibition was released by AMP. The ligands had no effect on the purified transaminase from pig heart. At lower temperatures AMP inhibited transaminase activity but this inhibition was reversed by a desensitizing agent, urea. Results of this type suggest that protein-protein interactions between nonidentical subunits of enzyme aggregates may occur under physiological conditions and represent a form of interlocking metabolic control between separate pathways.
Sigmoidal response curves indicating cooperative behavior have been observed with systems more complex than soluble enzyme aggregates.
For example, it has been reported (117) that the relationship between the displacement to longer wavelengths of the a absorbance band of ferrocytochrome b of submitochondrial particles by antimycin A and the inhibitor concentration displays sigmoidal behavior. Also, the amount of inhibition of succinate-cytochrome c reductase by antimycin shows a sigmoidal response when activity is plotted as a function of the inhibitor concentration. Cholate, an agent that disorients and disaggregates the respiratory membrane, causes these effects to approach a linear relation
ship. In the case of phase-inhomogeneous, membranous, or highly or
ganized macromolecular structures, results of this type become extremely difficult to interpret in terms of the simple allosteric models.
Indeed, it has been suggested (11$) that many macromolecular processes other than metabolic enzymic catalysis may be under a type of allosteric control. Since protein conformational changes imply physical movement or translocation of peptide sequences and associated bound ligands, Hill proposed (11$) that active transport, muscle contraction, and ribosomal translocation may utilize a common allosteric mechanism in which ATP and GTP have a dual role, providing energy through hydrolysis and acting as allosteric effectors.
Throughout the literature of allosteric effects, the primary action of effector ligands is assumed to be exerted through changes in three-dimen
sional protein structure. In some cases, indirect experimental proof for this assumption has been reported, as mentioned previously. Sedi
mentation coefficients, electronic absorbance and fluorescence spectra, electrophoretic mobility, and other probes of three-dimensional structure all point to conformational changes induced by ligands. However, the mode of binding of ligands remains in question in practically all cases.
Presumably short- or medium-range forces such as charge transfer, van der Waals, or hydrogen bonding (or even catalyzed covalent bonding) could all provide the triggering energy for macromolecular rearrange
ments. Long-range ionic bonds have been implicated in the case of
phosphorylase b interactions with phosphates. Since most allosteric ligands are charged molecules, ionic bonding may certainly be implicated.
Ionic bonding and electrostatic effects associated with charged groups may operate over greater distances (up to 10 A) than other types of bonding. Direct effects of bound charged ligands have been suggested that would not require any structural change to occur. Epstein (US) has pointed out that many enzyme active centers exist in relatively nonpolar regions in which electrostatic interactions would have larger free energies than in more polar environments. Many studies of the de
pendence on pH of the action of heterotropic ligands in inducing or en
hancing substrate cooperativity point to the importance of the electro
static charge of the protein and of the ligands.
The questions of the types of ligand bonding and the mechanism of cooperative interactions may be elucidated in some cases by determining the topography of the specific regulatory binding sites. Final proof of stable configurational states can perhaps be obtained only in the most favorable cases in which complete structural analysis of native and modified enzymes can be accomplished.
ACKNOWLEDGMENT
The author thanks Mr. Barry R . Hoffman and Mr. W y m a n C. Adams for assistance in preparing the manuscript.
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