Günzler V, Brocks D, Henke S, Myllylä R, Geiger R, Kivirikko K I
Department of Medical Biochemistry, University of Oulu, Finland.
J Biol Chem. 1988 Dec 25;263(36):19498-504.
Peptides containing the unphysiological amino acid 5-oxaproline (Opr) in the sequence R1-Xaa-Opr-Gly-OR2 were found to inactivate prolyl 4-hydroxylase from chick and human origins. Of the substances investigated, compounds with aromatic substituents R1 and R2 were particularly effective when compared with those with an aliphatic group or without a C-terminal blocking group. Both affinity of the individual peptides for the enzyme and partition ratio contributed to the differences in efficiency. Benzylcarbonyl-Phe-Opr-Gly-benzyl ester was the most effective substance tested, its concentration giving 50% inactivation in 1 h being 0.8 microM. Inactivation was only observed in the presence of 2-oxoglutarate and Fe2+. The Opr peptides enhanced the decarboxylation of 2-oxoglutarate by prolyl 4-hydroxylase, the Vmax values obtained with the individual peptides being positively correlated with their inactivating efficiency. Inactivation was prevented by high concentrations of peptide substrate and ascorbate. Lineweaver-Burk kinetics experiments suggested noncompetitive inhibition with respect to peptide substrate and ascorbate. Lysyl hydroxylase was not affected by Opr peptides in concentrations of up to 1.5 mM in either the presence or absence of prolyl 4-hydroxylase. The results suggest that the oxaproline compounds are specific syncatalytic inactivators of prolyl 4-hydroxylase.
序列为R1-Xaa-5-氧代脯氨酸（Opr）-Gly-OR2的含非生理性氨基酸5-氧代脯氨酸（Opr）的肽可使源自鸡和人的脯氨酰4-羟化酶失活。在所研究的物质中，与具有脂肪族基团或没有C端封闭基团的化合物相比，具有芳香族取代基R1和R2的化合物特别有效。各个肽对该酶的亲和力和分配比都导致了效率上的差异。苄羰基-Phe-Opr-Gly-苄酯是所测试的最有效的物质，其浓度在1小时内使50%失活时为0.8微摩尔。仅在存在2-氧代戊二酸和Fe2+的情况下才观察到失活。Opr肽增强了脯氨酰4-羟化酶对2-氧代戊二酸的脱羧作用，各个肽获得的Vmax值与其失活效率呈正相关。高浓度的肽底物和抗坏血酸可防止失活。Lineweaver-Burk动力学实验表明，相对于肽底物和抗坏血酸存在非竞争性抑制。在存在或不存在脯氨酰4-羟化酶的情况下，赖氨酰羟化酶在浓度高达1.5毫摩尔时均不受Opr肽的影响。结果表明，氧代脯氨酸化合物是脯氨酰4-羟化酶的特异性同步催化失活剂。
