The mussel foot secretes a variety of unusual hydroxyprolinecontaining collagenous and noncollagenous proteins. Prolyl4-hydroxylase acting on one or more of the secreted proteins was isolated from the foot by using conventional gel filtration and ion exchange chromatography. M, of the intact enzyme was 230,000 (olZ&J composed of two subunits with M, of 60,000 (a ) and 57,000 Q3) as estimated by HPLC gel filtration and SDS-PAGE. The enzyme utilized (Pro-Pro-GlyIlo as a substrate with a n apparent K, value of 0.17 mM.
Cofactors and inhibitors were very similar to animal, plant, and microbial prolyl hydroxylases previously described. The enzyme had a relatively sharp pH optimum in the range of 7.8-8.3 and the hydroxyproline formed increased in proportion to the rise in the temperature between 5 and 20ยฐC. No detectable hydroxylation occurred with poly-L-proline or the unhydroxylated decapeptide analog (Ala-Lys-Pro-Ser-Tyr-Pro-Pro-Thr-Tyr-Lys) of the polyphenolic protein.
Kinetic studies, however, revealed that the mussel prolyl 4-hydroxylase was competitively inhibited by poly-L-proline and uncompetitively inhibited by the decapeptide. These results suggest that the decapeptide binds the enzymesubstrate i.e. (Pro-Pro-Gly),o complex. It is not yet clear whether this enzyme acts exclusively on collagenous substrates or whether its catalytic purview extends as well to the polyphenolic protein.