AME 75:271-281 (2015)  -  DOI:

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

1Department of Earth and Planetary Sciences, University of Tennessee, 306 EPS Building, Knoxville, TN 37996, USA
2Department of Microbiology, University of Tennessee, M409 Walters Life Sciences, Knoxville, TN 37996, USA
3Department of Civil and Environmental Engineering, University of Tennessee, 325 John D. Tickle Building, Knoxville, TN 37996, USA
4Department of Biology, University of North Carolina at Chapel Hill, Coker Hall, CB #3280, Chapel Hill, NC 27599, USA
*Corresponding author:

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

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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.

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