The Structure and Enzyme-Coenzyme Relationship of Supernatant Aspartate Transaminase after Dye Sensitized Photooxidation

Abstract

Abstract Photooxidation of supernatant glutamate aspartate transaminase, limited to the stage during which much of the enzyme activity and only histidine residues are destroyed, does not induce gross protein structural changes as judged by ultracentrifugation, optical rotatory dispersion in the ultraviolet region, and microcomplement fixation techniques. Photooxidized apoenzyme has been shown to bind at least 90% as much coenzyme as the native enzyme and forms holopyridoxal or pyridoxamine enzyme which is very similar to the native enzyme in optical and absorption spectral properties in the visible region of the spectrum. The absorption spectrum of photooxidized apo- or holoenzyme has a new maximum at 325 mµ. Because of analogy to the absorption of the photooxidation product of a model compound, glycyl-histidyl-glycine, the new maximum in photooxidized transaminase has been assigned to the photoproduct of the altered histidines in the enzyme. Circular dichroism measurements of apoenzyme revealed positive ellipticity bands in the 280 to 300 mµ regions of the spectrum. They remain essentially unaltered after photooxidation. Binding of pyridoxal phosphate to this apoenzyme results in the appearance of the well known positive ellipticity bands in the visible region and formation of new negative ellipticity bands centered at 298 and 290 mµ in the aromatic region. Photooxidation eliminates the 290 mµ band from the pyridoxal form of the holoenzyme. The circular dichroism pattern of pyridoxamine phosphate bound to photooxidized transaminase is identical with that of native pyridoxamine holoenzyme only if this enzyme form is produced by transamination of photooxidized pyridoxal holoenzyme, not if formed by addition of pyridoxamine phosphate to photooxidized apoenzyme. Another physical anomaly resulting from photooxidation is the shift of the bound pyridoxal phosphate pK from 6.3 to 6.8. Since controlled photooxidation results in modification of only histidyl residues, this shift in pK may be due to the modification of a histidine in the steric vicinity of the pyridoxal phosphate at the active site, resulting in a localized perturbation of the environment of the coenzyme. The role of the histidyl residue at the active site cannot be associated with coenzyme binding.