PDF(Pubmed)
Abstract：
Human hyaluronidase 1 (HYAL1) and PH20 play vital roles in degrading hyaluronic acids through the substrate-assisted double displacement mechanism. While HYAL1, a lysosomal enzyme, functions optimally under acidic conditions, PH20, a sperm surface hyaluronidase, displays a broader pH range, from acidic to neutral. Our objective was to extend HYAL1\'s pH range towards neutral pH by introducing repulsive charge-charge interactions involving the catalytic Glu131, increasing its pKa as the proton donor. Substituting individual acidic residues in the β3-loop (S77D), β3\'-β3″ hairpin (T86D and P87E), and at Ala132 (A132D and A132E) enabled HYAL1 to demonstrate enzyme activity at pH 7, with the mutants S77D, P87E, and A132E showing the highest activity in the substrate gel assay. However, double and triple substitutions, including S77D/T86D/A132E as found in the PH20 configuration, did not result in enhanced activity compared to single substitutions. Conversely, PH20 mutants with non-acidic substitutions, such as D94S in the β3-loop and D103T in the β3\'-β3″ hairpin, significantly reduced activity within the pH range of 4 to 7. However, the PH20 mutant E149A, reciprocally substituted compared to A132E in HYAL1, exhibited activity similar to PH20 wild-type (WT) at pH 7. In a turbidimetric assay, HYAL1 mutants with single acidic substitutions exhibited activity similar to that of PH20 WT at pH 7. These results suggest that substituting acidic residues near Glu131 results in HYAL1 activity at neutral pH through electrostatic repulsion. This study highlights the significance of charge-charge interactions in both HYAL1 and PH20 in regulating the pH-dependent activity of hyaluronidases.
摘要：
人透明质酸酶1(HYAL1)和PH20通过底物辅助双置换机制在透明质酸降解中起重要作用。而HYAL1，一种溶酶体酶，在酸性条件下功能最佳，PH20，一种精子表面透明质酸酶，显示更宽的pH范围，从酸性到中性。我们的目标是通过引入涉及催化Glu131的排斥电荷-电荷相互作用，增加其作为质子供体的pKa，将HYAL1的pH范围扩展到中性pH。取代β3环（S77D）中的单个酸性残基，β3'-β3”发夹（T86D和P87E），在Ala132（A132D和A132E）使HYAL1在pH7时显示酶活性，突变体S77D，P87E,和A132E显示在底物凝胶测定中的最高活性。然而,双重和三重替换，包括PH20配置中的S77D/T86D/A132E，与单个替换相比，没有导致增强的活性。相反，具有非酸性取代的PH20突变体，如β3环中的D94S和β3'-β3″发夹中的D103T，在4至7的pH范围内显着降低活性。然而,PH20突变体E149A,与HYAL1中的A132E相比,相互取代,在pH7表现出与PH20野生型(WT)相似的活性。在比浊法中，具有单个酸性取代的HYAL1突变体在pH7下表现出与PH20WT相似的活性。这些结果表明，取代Glu131附近的酸性残基会通过静电排斥在中性pH下产生HYAL1活性。这项研究强调了HYAL1和PH20中电荷-电荷相互作用在调节透明质酸酶的pH依赖性活性中的重要性。
