The substrate recognition site of collagen proline hydroxylase: the hydroxylation of -X-Pro-Gly- sequences in bradykinin analogs and other peptides

A series of bradykinin (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg) analogs, in which one or more amino acid residues were substituted or added to the molecule, were tested as substrates of collagen proline hydroxylase. Analogs with substitutions at position 1 (lys¹-bradykinin), at position 2 (ala²-bradykinin), at position 6 (gly⁶-bradykinin) and position 1 and 9 (nitroarg^1,9-bradykinin) were hydroxylated by the enzyme. Analogs in which 1, 2, 3, or 9 amino acid residues had been added at the N-terminus of the molecule were also hydroxylated, and in general were more effective substrates than bradykinin itself. In contrast, modification of bradykinin by introducing substituents at position 3 (d-pro³-bradykinin, val³-bradykinin) or at position 4 (ala⁴-bradykinin, sar⁴-bradykinin, 5-aminoval⁴-bradykinin) yielded analogs which were not hydroxylated. Kinetic data for many of the active analogs are reported. A synthetic bradykinin-potentiating peptide (C) containing one -Pro-Pro-Gly-sequence could also act as a substrate, but the tetrapeptide Gly-Pro-Gly-Gly did not interact with the enzyme. It is concluded that the minimum sequence requirement for proline hydroxylation is an intact -X-Pro-Gly-triplet, and that the affinity of individual prolylcontaining sequences for the enzyme, and the rate at which they are hydroxylated are dependent on the adjacent amino acid residues and the identity of X.     

A rapid assay for collagen proline hydroxylase

A method for the assay of collagen proline hydroxylase is presented. Hydroxyproline-deficient protein, which serves as substrate, is prepared by incubating minces of chick embryo with 3,4-T-l-proline in the presence of α,α′-dipyridyl. Following extraction and partial purification, the substrate is incubated with proline hydroxylase, which converts some of the 3,4-T-l-proline residues to 3-T-l-hydroxyproline. The tritium atom which is released from carbon 4 equilibrates with water and the resulting tritiated water is assayed following vacuum distillation. It was shown that tritiated water release is entirely dependent on enzymic hydroxylation and that equivalent amounts of 3-T-l-hydroxyproline and tritiated water are formed. This method is much more rapid and accurate than previously described methods.     

Cofactor and substrate requirements of collagen proline hydroxylase

Enzyme fractions which catalyze the hydroxylation of peptidyl-proline to form peptidyl-hydroxyproline have been prepared from chick embryo, guinea-pig granuloma and fetal rat skin. In all preparations ferrous ion, ascorbic acid, α-ketoglutarate and molecular oxygen are required for activity. No compounds have been found which can replace ferrous ion and α-ketoglutarate. Reduced pteridines can partially replace ascorbic acid, but reduced pyridine nucleotides are inactive. The enzyme has been called collagen proline hydroxylase and has been tentatively classified as a mixed-function oxidase. A formulation of the reaction catalyzed by this enzyme has been proposed in which ascorbate functions as a reducing agent and α-ketoglutarate as a modifier of the enzyme.     

Inhibition of prolyl hydroxylase activity by bradykinin analogs containing a prolyl-like residue

A number of substituted bradykinin analogs were prepared in which the proline in position 3 was replaced by analogs of proline. All of the bradykinin analogs, with the exception of l-azetidine-2-carboxyl³-bradykinin showed significant ability to inhibit prolyl hydroxylase activity. Addition of an l-glutamyl residue to the amino terminus of 3,4-dehydro-l-prolyl³-bradykinin and trans-4-hydroxy-l-prolyl³-bradykinin resulted in competitive inhibitors of increased effectiveness with K_i, values approximately 10^?4m. One of the peptides, l-3,4-dehydro-l-prolyl³-bradykinin, appeared to serve as a substrate for prolyl hydroxylase.     

Substrate specificity of the paraffin hydroxylase ofPseudomonas aeruginosa

The paraffin hydroxylating enzyme system isolated fromPseudomonas aeruginosa (strain 473) grown onn-heptane has been studied with respect to its substrate specificity. It has been found that cell-free extracts of this organism can hydroxylate a wide variety of hydrocarbons. In order to function as substrates, molecules should be able to assume a more or less planar conformation. The compounds used in this study can be divided into three classes: (a) alkanes, (b) alkylbenzenes and (c) (alkyl)cycloalkanes. In each of these groups hydroxylation occurs at a specific site in the molecule. Noteworthy is the hydroxylation of isopropyl groups at a methyl carbon and that of monoalkylcyclohexanes at the 4-transposition.From the geometry of the substrate and non-substrate molecules the conclusion was drawn that the active centre of the hydroxylase is a cleft in the enzyme surface of about 5 Å wide and 8 Å deep.We gratefully acknowledge the skillfull assistance of Miss W. T. Hempel.     

Constancy of proline hydroxylation in collagen synthesized at different temperatures by poikilotherms.

Previous experiments have suggested that the body temperature of an organism may in part regulate the hydroxyproline content of its collagen. In order to test this hypothesis, the hydroxyproline content of the collagen synthesized at different temperatures by fish cells in culture and by intact amphibian larvae was measured. High levels of prolyl hydroxylation were found at both low and high temperatures, which eliminates the possibility that temperature can directly modulate prolyl hydroxylation in vivo. Synthesis and hydroxylation of collagen appear to be coordinated so that unstable underhydroxylated molecules are not produced under normal circumstances.     

The role of hydroxylation of proline in the antigenicity of basement membrane collagen.

Rabbit antibodies to bovine basement membrane collagen were used to compare the antigenic determinants of rat parietal yolk sac basement membrane [14C]procollagen with [14C]protocollagen. Basement membrane [14C]protocollagen was found to be less antigenic than basement membrane [14C]procollagen. Hydroxylation of basement membrane [14C]protocollagen, either intracellularly or in vitro with protocollagen prolyl hydroxylase, resulted in restoration of antigenicity. The difference in antigenicity observed between basement membrane [14C]procollagen and basement membrane [14C]protocollagen appeared to depend primarily upon the presence of hydroxyproline in the collagen molecule. Glucosylgalactosylhydroxylysine was found to be unimportant for antigenicity.     

In vitro hydroxylation of proline in the collagen of the cysticercus of Taenia solium

1. In vitro hydroxylation of proline in cysticercus collagen was carried out using two different vertebrate enzymes. 2. Chick embryo enzyme is more active on cysticercus collagen than new-born rabbit enzyme. 3. Hydroxylation of cysticercus collagen is more efficient at 40 than at 37 degrees C. 4. No prolyl hydroxylase activity was detected in cysticercus extracts. 5. Collagen from the adult tapeworms Taenia solium and T. saginata lack hydroxyproline. 6. SLS crystallites from T. solium and T. saginata collagen show the same band pattern as cysticercus collagen.