Decarboxylation of alpha-ketoglutarate coupled to collagen proline hydroxylase.

The collagen proline hyd.roxylase system (also referred to as protocollagen hydroxylase), which converts selected proline residues to hydroxyproline in polypeptide precursors of collagen, has now been purified from a variety of animal tissues.1-4 M\/Iost purified preparations have an absolute requiremenlt for Fe++ and for a reducing agent, which can be ascorbic acid, other enediols, or tetrahydropteridines. An additional requirement discovered by Hutton et al.1' is a-ketoglutarate. This keto acid cannot be replaced by pyruvate, oxaloacetate, or related compounds and is an absolute requirement for proline hydroxylation. Removal of the keto acid by incubation with crystalline glutamic dehydrogenase, NH4+, and reduced nicotinamide-adeniine dinucleotide (NADH) leads to total inactivation of crude homogenates; activity is restored by the addition of aketoglutarate. Earlier attempts in this laboratory to determine whether a-ketoglutarate was consumed during the course of the hydroxylation reaction were inconclusive because the chick-embryo enzyme preparations used contained other enzymes that metabolized a-ketogluftarate.1 More recently, we have been able to obtain preparations from fetal, rat; skin that are far more active with respect to proline hydroxylation and that do not contain competing enzymes. With these preparations, it has now been possible to show a substrate-dependent and stoichiometric decarboxylation of a--ketoglutarate coupled to the hydroxylation of peptidyl proline residues. Ml/aterials and M11ethods. -Both H(3,4-Hl3-Pro-Gly-Pro),OH (mol wt 4100; spee. act. 0.9 mec/nmg) and poly-L-proline 11 (mol wt 9000) were gifts from Drs. J. Kurtz and A. 13erger, Weizmanari I nstitute of S ciences, Rehovoth, Israel. We obtaiiied a-ketoglutarate5-C14, (9.33 me/mnmole) from Nuelear-Chicago Corp., and a-ketoglutarate-U-C14 (3.2 me/mmole) an-d a-ketoglutarate-1-C14 (11 me/mmole) from Calbiochem, Inc. These were diluted with nonradioactive a-ketogluitarate before use. Crystalline bovine liver L-glutamic acid dehydrogenase) crystalline bovine liver catalase, and NADH were all products of Sigma Chemical Company. NCS reagent (for trapping C02) was obtained from Nuclear-Chicago Corp., and crystalline bovine serum albumin from Pentex Corp. Collagen proline hydroxylase was prepared from skins of newborn rats. Approximately 300 gm of tissue were homnogenized in 2 vol of 0.25 M. sucrose containing 0.01 mM ethylenediaini,1etet-aacetate (E})TA) aid O.l OJmM dithiothreitol, and the homogenate was centrifuged at 27,000 X g. All solutions used in subsequent purificatioin contained the same coneentrations of ditliothieitol. TFwo vol of saturated ammonium sulfate at pH 7.0 were added to the supelrnatant fraetion, and after centrifugation, the precipitate was dissolved in sodium cacodylate buffer, pH 7.0, and treated with 0.64 mg streptomycin sulfate per mg protein to remove nucleic acids. After centrifugation for 1 hr at 68,000 X g, the supernatanit fluid was treated with nieutral ammonium sulfate, and the fraction that precipitated between 17%( and 39% saturation (12 and 27 gm/100 ml) was dissolved in 0.05 M cacodylate, pH 7.0, containing 0.2 11f NaCl. After dialysis against 4 liters of the same solution, the en-zyme (800 mg of protein) was charged onto an O-(diethyl-