Abstract
In the presence of substrate (phosphate or glucose-1-phosphate) and allosteric activator (AMP), differential spectra of an enzyme-substrate complex of phosphorylase b were obtained. By quantitative measurements of affinity constants, it was shown that the spectra are due to a Michaelis complex of the enzyme with the substrate. These data prove that pyridoxal-phosphate has an important role in the Michaelis complex, i.e. it binds the substrate in anionic form. It is shown that the origin of the differential spectrum is a slight shift of the absorption band of pyridoxal-phosphate due to the conversion of the pyridine cycle from non-protonated to the protonated form. The intensity of the differential spectrum is proportional to the derivative of the intensity of the main absorption band. In the presence of an allosteric inhibitor, the differential spectrum is suppressed. The dependence of its intensity on substrate concentration acquires a characteristic sigmoidal shape.
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