Are spontaneous conformational interconversions a molecular basis for long-period oscillations in enzyme activity?
An unconventional hypothesis to the molecular basis of enzyme rhythms is that the intrinsic physical instability of the protein molecules which, in an aqueous medium, tend to move continuously from one conformational state to another could lead, in the population of enzyme molecules, to sizeable long-period oscillations in affinity for substrate and sensitivity to ligands and regulatory effects. To investigate this hypothesis, malate dehydrogenase was extracted and purified from leaves of the plant Kalanchoe blossfeldiana. The enzyme solutions were maintained under constant conditions and sampled at regular intervals for up to 40 or 70 h for measurements of activity as a function of substrate concentration, Km for oxaloacetic acid and sensitivity to the action of 2,3-butanedione, a modifier of active site arginyl residues. The results show that continuous slow oscillations in the catalytic capacity of the enzyme occur in all the extracts checked, together with fluctuations in Km. Apparent circadian periodicities were observed in accordance with previous data established during long run (100 h) experiments. The saturation curves for substrate showed multiple kinetic functions, with various pronounced intermediary plateaus and "bumps" depending on the time of sampling. Variation in the response to the effect of butanedione indicated fluctuation in the accessibility to the active site. Taken together, the results suggest that, under constant conditions, the enzyme in solution shifts continuously and reversibly between different configurations. This was confirmed by parallel studies on the proton-NMR spectrum of water aggregates in the enzyme solution and proton exchange rates.(ABSTRACT TRUNCATED AT 250 WORDS)
Queiroz-Claret
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Institut de Physiologie Végétale, C.N.R.S., Gif-sur-Yvette, France.
Valon
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Queiroz
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D001665:Binding Sites; D002940:Circadian Rhythm; D004798:Enzymes / Q000378:metabolism*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008291:Malate Dehydrogenase / Q000378:metabolism*; D008962:Models, Theoretical; D010507:Periodicity; D010944:Plants / Q000201:enzymology*; D011487:Protein Conformation
D004798:Enzymes; D008291:Malate Dehydrogenase