Characterizing the Functional Role of Tyrosine 381 in the Activity of E. coli Aminopeptidase N (PepN) Through Mutagenesis

Abstract

Aminopeptidase N (PepN) from E. coli is an M1 class peptidase with broad specificity. PepN was over‐expressed, purified, and mechanistically characterized with L‐alanine‐p‐nitroanilide in an effort to understand the role of tyrosine 381. In an effort to characterize the functional roles of binding region components, this tyrosine was mutated to alanine, Y381A‐ PepN, and phenylalanine, Y381F‐ PepN. These mutants included a silent mutation, that is an addition or subtraction of an restriction enzyme site to easily identify successful mutagenesis. Successful mutants were over‐expressed, purified, and mechanistically characterized. Y381A‐PepN exhibited greater than 900‐fold loss of activity (kcat), while Y381F‐PepN maintained comparable activity, when compared to the wild‐type PepN. This suggests that Tyr381 has an essential mechanistic role in the function of PepN beyond the suggested role of hydrogen bond stabilization of substrate and product.Support or Funding InformationThis work was funded by Augustana College New Faculty Research Fund, Augustana College Research Foundation, and Augustana College Edwin Erickson Summer Research Award.Agarose gel showing silent mutation, addition of a NarI restriction site to Y381A‐PepN.Lanes from left to right: wild‐type, Y381A1‐PepN, Y381A2‐PepN. All samples were digested with NarI for 1 hour at 37°C. Y381A1 shows an unsuccessful mutation (lack of alteration to restriction digestion map). Y381A2 shows an successful mutation (an alteration to restriction digestion map). New restriction digest bands due to the silent mutation are below main band.Figure 1