Function Of Carboxypeptidase Research Articles

Role of carbohydrate moiety in carboxypeptidase Y: structural study of mutant enzyme lacking carbohydrate moiety.

To study the roles of the carbohydrate moiety in the function of carboxypeptidase Y, asparagine residues at 13, 87, 168, and 368, the four-consensus N-linked glycosylation sites, were altered to alanine with site-directed mutagenesis. The mutant enzyme of 51 kDa completely lost the carbohydrate moiety which was present in the 61-kDa wild-type enzyme. Structural studies of the mutant enzyme showed that it maintained the native-like structure; hydrolytic activity, and substrate specificity of the mutant enzyme analogous to those of the wild-type enzyme. Susceptibility of the mutant enzyme toward proteolysis and pressure denaturation was reduced by 10-20%. It is concluded that the carbohydrate moiety functions to maintain the structural integrity of the enzyme under stressed.

Effects of pH on the structure and function of carboxypeptidase A: crystallographic studies.

High-resolution crystal structures are described for carboxypeptidase A (EC 3.4.17.1) in crystals grown at pH 8.5, 9.0, and 9.5 and compared with the structure at pH 7.5. The comparison shows that in the pH range of 7.5-9.5 the enzyme structure is practically unchanged, and, most importantly, that the flexible side chain of Tyr-248 remains exclusively in the "up" position, away from the Zn atom, throughout the pH range. There is no evidence for binding of Tyr-248 to Zn at any of these pH values. We conclude that the interaction of Tyr-248 with Zn is not an essential part of the mechanism of carboxypeptidase A and that its occurrence is an artifact of chemical modification of Tyr-248. It is also suggested that Tyr-248 is not uniquely associated with the observed high pK of the enzymatic hydrolysis.

Structure and function of carboxypeptidase A alpha in supercooled water.

The spectral and enzymatic characteristics of chromophoric derivatives of carboxypeptidase A alpha (EC 3.4.17.1) have been examined at subzero temperatures in supercooled water-in-oil emulsions. Substrate and temperature dependencies of enzyme kinetics indicated the existence of a solution-like enzyme phase that greatly extends the temperature range (greater than 60 degrees C) over which the activity of this enzyme can be measured. The emulsion spectra were virtually identical to those of solutions over a wide range of temperatures. Subzero temperatures (less than -10 degrees C) may induce changes of enzyme conformation but not of geometry at the site of the metal atom, nor do they adversely affect activity at any of the temperatures studied. Both structure and function of carboxypeptidase A alpha can be examined in supercooled water under identical reaction conditions.

Effect of Ultraviolet Irradiation on Composition and Function of Carboxypeptidase A*

Effect of Ultraviolet Irradiation on Composition and Function of Carboxypeptidase A^*