Endopeptidase 24.16B: A new variant of endopeptidase 24.16

A peptidase, isolated from rat testes, is inhibited by 1 MM o-phenanthroline, 1 μM N-(1-R,S)-carboxyl-3-phenylpropyl)-Ala-Ala-Phe-p-aminobenzoate, and 6 mM Pro-Ile, properties similar to those ascribed to endopeptidase 24.16. The enzyme hydrolyzes dynorphin A-8, neurotensin 1-13, angiotensin I, and substance P. Kinetic analysis of a series of angiotensin I analogs showed that substitutions at P-1, P-1′, or P-2′ had little effect on K_m or k_cat. Variation of peptide size with a series of dynorphin A peptides showed chain length to be significant. The peptidase cleaved dynorphin A-8 at both Leu⁵-Arg⁶ and Arg⁶-Arg⁷, and neurotensin 1-13 at Pro¹⁰-Tyr¹¹ and Arg⁸-Arg⁹. In contrast, rat endopeptidase 24.16 cleaves dynorphin A-8 at Gly⁴-Leu⁵ and Leu⁵-Arg⁶, and neurotensin 1-13 only at Pro¹⁰-Tyr¹¹. These findings, as well as the observation that endopeptidase 24.16 exhibits a considerably higher affinity for Pro-Ile, K_i = 90 μM, indicates the peptidase isolated in this study is related to, but distinct from, rat endopeptidase 24.16. We propose that this new endopeptidase be referred to as endopeptidase 24.16B, while the originally described enzyme be referred to as endopeptidase 24.16A.