Arsanilazotyrosine 248 carboxypeptidase

Although many of these general features are correct, these mechanistic conclusions are based upon the assumption that the properties of the enzyme in solution are conserved on crystallization. It appears from Raman studies on arsanilazotyrosine-248 carboxypeptidase that the enzyme exists in solution in a number of different forms.509 It is not certain that the form which crystallizes out is the kinetically active species. Indeed there is evidence that the kinetics of carboxypeptidase in solution differ from those of the enzyme crystals, with the crystalline enzyme being 1000-fold less active with some substrates.510 The interaction of Gly-L-tyr with Mn carboxypeptidase A, studied by 1H NMR techniques, does not involve coordination of the carboxyl group of the substrate, and may well represent a different conformation from the one studied by crystallographic techniques.511 Several conformational forms of the Cd11 carboxypeptidase A have been found in solution, while the enzyme exists in a different form in the crystal state.312... 

In their classic study they were able to modify specifically tyrosine 248 in the zinc metalloenzyme, carboxypeptidase A, to give the azotyrosine derivative, arsanilazotyrosine 248 carboxypeptidase A (AA-CPA-Zn), shown in Figure 3A. The native Zn is shown explicitly in order to differentiate it from externally incorporated Co as will be discussed. They found that at intermediate pH s, where the enzyme exhibits maximal activity, the azotyrosine is chelated to the intrinsically bound active site zinc. A distinct red color is associated with zinc chelation in contrast to the yellow and orange colors of the enzyme due to the presence of the free azophenol (low pH) and azophenolate (high pH), respectively (7). 

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