AME 75:271-281 (2015) - DOI: https://doi.org/10.3354/ame01755
Substrate specificity of aquatic extracellular peptidases assessed by competitive inhibition assays using synthetic substrates
Andrew D. Steen1,*, Jasmine P. Vazin2, Shane M. Hagen3, Katherine H. Mulligan2,4, Steven W. Wilhelm2
ABSTRACT: The identities and biochemical properties of extracellular enzymes present in natural environments are poorly constrained. We used a series of competitive inhibition experiments with samples from a freshwater environment (the Tennessee River at Knoxville, TN, USA) and a marine environment (Bogue Sound, NC, USA) to characterize the range of substrate specificities of naturally occurring enzymes that hydrolyze L-leucine 7-amido-4-methylcoumarin (Leu-AMC), L‑proline-AMC (Pro-AMC), and L-arginine-AMC (Arg-AMC)—putative substrates for leucyl-aminopeptidase, prolyl-aminopeptidase, and arginyl-aminopeptidase, respectively. Extracellular peptidases which hydrolyzed Arg-AMC and Leu-AMC demonstrated affinity for up to 8 other amino acids, whereas those hydrolyzing Pro-AMC in the Tennessee River, and Arg-AMC at Bogue Sound, were more specific to proline and arginine, respectively. Patterns of substrate affinity showed that Leu-AMC (at both sampling sites) and Arg-AMC (at Bogue Sound) were primarily hydrolyzed by enzymes other than leucyl-aminopeptidase and arginyl-aminopeptidase, respectively. The set of naturally occurring peptidases in both environments showed greater affinity towards a subset of amino acids. These amino acids were on average larger, yielded more free energy from oxidation to CO2, and tended to be depleted in aged organic matter. These relationships indicate that pathways of amino acid diagenesis are at least partially controlled by the substrate specificities of the peptidases involved in protein degradation.
KEY WORDS: Extracellular enzymes · Peptidases · Protein degradation · Heterotrophy · Amino acids
Full text in pdf format  | Cite this article as: Steen AD, Vazin JP, Hagen SM, Mulligan KH, Wilhelm SW
(2015) Substrate specificity of aquatic extracellular peptidases assessed by competitive inhibition assays using synthetic substrates. Aquat Microb Ecol 75:271-281. https://doi.org/10.3354/ame01755
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