Comparing extracellular enzymatic hydrolysis between plain peptides and their corresponding analogs in the northern Gulf of Mexico Mississippi River plume

Peptide analogs, such as leucine-methylcoumarinylamide (Leu-MCA) and Lucifer yellow anhydride-tetraalanine (LYA-Ala4), are often used as proxies to quantify extracellular peptidase activities, but how accurately these analogs represent natural peptides remains unclear. Here we compared hydrolysis rates of tetrapeptides alanine-valine-phenylalanine-alanine (AVFA) and tetraalanine (Ala4) with their corresponding LYA analogs along a salinity gradient in the northern Gulf of Mexico Mississippi River plume. Hydrolysis rates were generally similar, but not always, between natural peptides and their analogs in the plume water. For example, hydrolysis rates were similar between LYA-AVFA and AVFA at all stations except one. In contrast, hydrolysis rates of LYA-Ala4 were significantly higher than those of Ala4 at low-salinity (18-19) stations, but significantly lower at one high-salinity (36) station. These results suggest that relative activities of different types of peptidases, measured by the analogs or peptides, depended on biological and chemical properties at each station, such as abundance of certain “opportuni-trophic” bacteria, including Flavobacterium, Ruegeria and Roseobacter. The produced fragments and amino acids from peptide hydrolysis implied further that the hydrolysis pathway depended on specific peptide substrate, and that the LYA tag affected the hydrolysis pathway. Taken together, this study validates the technique of using LYA analogs to study extracellular enzymatic activities, particularly the overall hydrolysis rate, and provides insights into the patterns of extracellular enzymatic activities in estuarine environments.