A micellar model for the pyridoxal 5′-phosphate site of glycogen phosphorylase

Viviane Gani, Abraham Kupfer, Shmuel Shaltiel

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Abstract

Mixed micelles composed of hexadecyltri-methylammonium bromide and dodecylamine hydrochloride were found to be good models for the site accommodating pyridoxal 5′-phosphate (pyridoxal-5′-P) in glycogen phosphorylase. At neutral pH, pyridoxal-5′-P becomes entrapped by such micelles (as shown by gel filtration) and reacts in stoichiometric ratios with the primary amine to yield a Schiff base derivative embedded in a hydrophobic microenvironment. The resulting pyridoxal-5′-P micelles simulate the enzyme in their absorption spectrum (maxima at 333 nm (major) and at ∼415 nm (minor)) and in their fluorescence characteristics (excitation maxima at 335 and ∼415 nm, emission maximum at ∼550 nm). These pyridoxal-5′-P micelles can be reduced with NaBH4 with concomitant disappearance of the 333-nm band to yield a product with an absorption maximum at 288 nm similar to the absorption of NaBH4-reduced phosphorylase at neutral pH. Removal of pyridoxal-5′-P from these micelles ("resolution") can be achieved by reaction with cysteine and gel filtration. This process resembles the resolution of "deformed" phosphorylase in that it takes place through an intermediate step whereby cysteine penetrates the micelle and reacts within the micelle with the entrapped pyridoxal-5′-P Schiff base. Unlike previous models for the vitamin B6 site of phosphorylase, which consisted of Schiff base derivatives of the cofactor in nonaqueous solvents, the micellar model described here (like the functioning enzyme molecule) puts the pyridoxal-5′-P Schiff base in a hydrophobic microenvironment within an aqueous milieu, which can accommodate the water-soluble natural substrates of the enzyme.

Original languageEnglish (US)
Pages (from-to)1294-1300
Number of pages7
JournalBiochemistry®
Volume17
Issue number7
StatePublished - 1978
Externally publishedYes

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ASJC Scopus subject areas

  • Biochemistry

Cite this

Gani, V., Kupfer, A., & Shaltiel, S. (1978). A micellar model for the pyridoxal 5′-phosphate site of glycogen phosphorylase. Biochemistry®, 17(7), 1294-1300.