The structure and enzyme-coenzyme relationship of supernatant aspartate transaminase after dye sensitized photooxidation.

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 apoor holoenzyme has a new maximum at 325 mp. 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 rnp 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 rnp in the aromatic region. Photooxidation eliminates the 290 rnp 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. Photooxidation of proteins in the presence of dyes can result in alteration of histidyl, tryptophyl, methionyl, tyrosyl, and cysteinyl residues (3, 4). However, photooxidation of soluble pig heart aspartate transaminase (EC 2.6.1.1) in the presence of either methylene blue or rose Bengal results in the alteration of only histidyl residues, with a concomitant loss of enzymatic activity (5). Since during photooxidation the first order rate constant for the alteration of rapidly oxidized histidyl residues is identical with the first order constant for the loss of enzymatic activity, it has previously been concluded that loss of catalytic activity was due to the specific alteration of one of the transaminase’s histidyl residues (5). This histidine need not necessarily be at the active site since the alteration of these protein amino acid residues could result in inactivation by changing the basic enzyme structure needed for maintenance of a catalytically efficient conformation. Indeed, methylene blue-sensitized photooxidation of dehydrogenases has been shown to cause inactivation because of gross changes in tertiary structure (6). On the other hand, this histidine could be located at the active site and function directly or indirectly in catalysis. Histidine residues have been implicated, for example, in the catalytic mechanism of rabbit muscle aldolase (7). It is important to distinguish between these possibilities if the role of this residue is to be understood. We have, therefore, carried out a series of studies on the structure of the photooxidized enzyme with spectral, optical, and immunochemical techniques to determine whether there are detectable differences between the structure of this enzyme and the native enzyme. Both protein structure and the enzyme-coenzyme relationship have been investigated in this work. The accompanying paper (8) deals with the assignment of a function for the active site histidyl residue in the mechanism of transamination.