[76] Spin-labeled vitamin B6 derivatives: Synthesis and interaction with aspartate aminotransferase

Spin-labeled analogs of coenzymes, substrates, and prosthetic groups have proved to be a useful tool for elucidating structure and conformational changes in proteins. This chapter describes the synthesis of spin-labeled vitamin B6 derivatives and their interaction with aspartate aminotransferase. The iminoxyl group of spin-labeled derivatives has been used not only as a “reporter” group but also as a specific oxidant of the cysteine residue. The chapter describes the method in which the paramagnetic analogs of pyridoxal 5'-phosphate (PLP) and pyridoxamine 5'-phosphate (PMP) are applied for studying AAT. The apoenzyme of AAT (apo-AAT) is stable enough and capable of binding an analog of the coenzyme. This results in a typical maximum in the circular dichroism (CD) spectrum, the appearance of which should be attributed to the asymmetrical surroundings of the chromophore. The interaction of the apo-AAT with certain paramagnetic analogs is also accompanied by a characteristic Cotton effect in the absorption band for analogs.