Gacesa P, Savitsky M J, Dodgson K S, Olavesen A H
Biochim Biophys Acta. 1981 Oct 13;661(2):205-12. doi: 10.1016/0005-2744(81)90005-x.
Purified bovine testicular hyaluronidase (hyaluronate 4-glycanohydrolase, EC 3.2.1.35) was inactivated by butane-2,3-dione in either borate or Hepes buffer, pH 8.3. The presence of borate enhanced the inactivation process which followed pseudo-first-order kinetics with a calculated second-order rate constant of 13.54M-1 min-1. Using kinetic data it was estimated that the modification of 1 mol arginine per mol enzyme was sufficient for inactivation to occur, whereas amino acid analysis indicated that 4 mol arginine had been modified. The inactivation process was partially prevented by using either competitive inhibitors or substrates of the enzyme, thus indicating that the essential arginine residue is close to the active site of hyaluronidase. A full kinetic analysis of the enzyme with either hyaluronic acid or chondroitin 6-sulphate as substrate showed that the activity of hyaluronidase was uncompetitively activated by either protons or NaCl. The product obtained by reduction of the corboxyl groups of hyaluronic acid to the corresponding alcohol groups was a competitive inhibitor. The possibility that the microenvironment of hyaluronic acid was responsible for the observed kinetic effects of pH and ionic strength was dispelled. It is concluded that these data are compatible with a mechanism that involves anionic interaction between a carboxyl group on the substrate and an arginine residue on the enzyme.
纯化的牛睾丸透明质酸酶（透明质酸4-聚糖水解酶，EC 3.2.1.35）在pH 8.3的硼酸盐或Hepes缓冲液中被丁二酮灭活。硼酸盐的存在增强了灭活过程，该过程遵循假一级动力学，计算得到的二级速率常数为13.54M-1 min-1。利用动力学数据估计，每摩尔酶修饰1摩尔精氨酸就足以导致灭活，而氨基酸分析表明有4摩尔精氨酸被修饰。使用该酶的竞争性抑制剂或底物可部分阻止灭活过程，因此表明必需的精氨酸残基靠近透明质酸酶的活性位点。以透明质酸或硫酸软骨素6-硫酸盐为底物对该酶进行的完整动力学分析表明，透明质酸酶的活性被质子或NaCl非竞争性激活。通过将透明质酸的羧基还原为相应的醇基而得到的产物是一种竞争性抑制剂。透明质酸的微环境导致观察到的pH和离子强度动力学效应的可能性被排除。结论是，这些数据与一种涉及底物上的羧基与酶上的精氨酸残基之间阴离子相互作用的机制相符。
