Purified L-aminopeptidase

Tabie 2 Substrate Specificity of the Purified L-Aminopeptidase from P. putida ATCC 12633... 

Racemic a-amino amides and a-hydroxy amides have been hydrolyzed enantio-selectively by amidases. Both L-selective and o-selective amidases are known. For example, a purified L-selective amidase from Ochrobactrum anthropi combines a very broad substrate specificity with a high enantioselectivity on a-hydrogen and a,a-disubstituted a-amino acid amides, a-hydroxyacid amides, and a-N-hydroxya-mino acid amides [102]. A racemase (a-amino-e-caprolactam racemase, EC 5.1.1.15) converts the o-aminopeptidase-catalyzed hydrolysis of a-amino acid amides into a DKR (Figure 6.38) [103]. 

P. E. Ward, I. F. Benter, L. Dick, S. Wilk, Metabolism of Vasoactive Peptides by Plasma and Purified Renal Aminopeptidase M Biochem. Pharmacol. 1990, 40, 1725 — 1732. 

Characterization of the purified protein by nondenaturing gel filtration reveals a native molecular mass of approximately 400 kDa. SDS-PAGE analysis of the pure enzyme shows a single band with a molecular mass of about 52 kDa. This indicates that the l-aminopeptidase most probably is a homo-octameric enzyme. The isoelectric point of the protein is estimated as pH 10.5 using an isoelectric focusing (lEF) 3-9 Ph tgel with an expanded pH range. 

The L-aminopeptidase from P. putida displays clear amide-hydrolyzing activity between pH 7 and 11, with the highest activity at pH 9.0-9.5. Furthermore, the enzyme has maximum activity at 40°C (Fig. 2). The thermal stability of this enzyme in purified form is rather limited after a 60-min incubation period at 30 C, only 65% of the initial activity remains. Addition of substrate, however, dramatically enhances the thermal stability (60 min at 30°C, 100% activity left). 

L-Alanine aminopeptidase has been purified from human kidney. The glycoprotein (mol. wt. 2.36 x 10 ) contains 21% carbohydrate, including residues of 2-amino-2-deoxy-D-glucose, jV-acetylneuraminic acid, and hexose. 

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