The molecular basis of the density increase of phenylalanine ammonia-lyase, extracted from the cotyledons of mustard (Sinapis alba) incubated on deuterium oxide

Abstract

Abstract Previous studies of deuterium labelling of proteins have measured shifts in buoyant density and inferred the incorporation of deuterium into the free amino acids. This paper reports the use of combined gas-liquid chromatography-mass spectrometry to measure the incorporation of deuterium into the free amino acids of Sinapis alba cotyledons, from which the expected density shift of phenylalanine ammonia-lyase (EC 4.3.1.5) has been calculated and compared with that measured by isopycnic density-gradient centrifugation. The quantitative results show that most of the amino acids entering phenylalanine ammonia-lyase are only deuterated to a small extent and pass from storage protein to phenylalamine ammonia-lyase virtually without engaging in enzyme-catalysed exchange reactions. Most of the deuterium incorporated into phenylalanine ammonia-lyase is transferred with the amino acids alanine (approx. 32%), glutamate (approx. 21%), and glutamine (approx. 16%). Possible reasons for the discrepancy between the calculated and observed shift in buoyant density of phenylalanine ammonia-lyase are discussed.