Formation of Enzyme-Substrate Complexes with Protocollagen Proline Hydroxylase and Large Polypeptide Substrates

摘要: Abstract Cartilage from chick embryos was incubated with 14C-proline and puromycin in order to prepare peptides which were substrates for the synthesis of 14C-hydroxyproline protocollagen proline hydroxylase. Stable enzyme-substrate complexes recovered when substrate mixed enzyme mixtures examined by gel filtration. Although ascorbate, α-ketoglutarate, ferrous iron are required catalytic activity, formation did not require addition cofactors or cosubstrates dialyzed enzyme. Formation reduced but prevented EDTA concentrations inhibited activity. Under conditions used, isolated readily saturated substrate, it difficult saturate With high relative initial velocity reaction greater than observed complexes, suggesting that 1 molecule polypeptide could bind more The equilibrium free since a competing produced dissociation rapid inhibition hydroxylation substrate. Most still under over-all concentration about 100 pm polypeptide. Since appeared be these conditions, results suggested constant expressed terms molar polypeptides probably less pm. affinity decreased after partial After one-sixth available converted 14C-hydroxyproline, no longer possible demonstrate filtration column. Previous kinetic measurements demonstrated has higher large small ones same structure, single encounter between involves segment present indicate multiple encounters necessary complete molecular weights up 100,000. decreases introduction final few hydroxyl groups into apparently does proceed as first groups.