α-L-Iduronidase ( IDUA ) 為一種溶小體酵素，主要代謝 heparan sulfate 及 dermatan sulfate 黏多醣，當酵素活性缺失或顯著降低，將造成黏多醣在體內廣泛儲積，導致體染色體隱性遺傳的第一型黏多醣儲積症 ( 簡稱 MPS I )。IDUA 基因已被分離並定序，根據國外 MPS I 患者的 IDUA 基因突變分析，發現 IDUA 基因的突變呈現高度異質性，並有多種多型性存在。 本論文主要探討台灣 MPS I 患者的 IDUA 基因突變 ( T364M、M1I+(54-100、Y343X、R619G ) 及多型性 ( R105Q、A361T、V454I ) 對 IDUA 基因表現的影響，及 IDUA 酵素的動力學特性。含突變 T364M 的 IDUA 重組質體轉移細胞株所表現的 IDUA 酵素活性、IDUA mRNA 量分別為野生型的 1.2 %、32 %，但未偵測到 IDUA 蛋白質，顯示突變 T364M 影響 IDUA mRNA 及蛋白質的穩定性，導致 IDUA 酵素活性降低。含突變 M1I+(54-100、Y343X、R619G 的重組質體轉移細胞株所表現的 IDUA 酵素活性分別為野生型的 10.5 %、4.5 %、4.9 %，IDUA mRNA 量則分別為野生型的 124 %、94 %、80 %，顯示此三個突變的影響主要在蛋白質層面。突變 M1I+(54-100 造成包含 signal peptide 的 N 端 132 個胺基酸的缺失，導致 IDUA 酵素活性降低且無法進入溶小體；突變 Y343X 造成包含一個糖化位置 ( Asn-336 ) 的第 325-343 個胺基酸的缺失，而且其轉移細胞株內未偵測到 IDUA 蛋白質，顯示突變 Y343X 影響 IDUA 蛋白質的運輸及穩定性，導致 IDUA 酵素活性降低；突變 R619G 的轉移細胞株可偵測到 IDUA 蛋白質，約為野生型的 52 %，因此突變 R619G 主要影響 IDUA 蛋白質的結構，導致 IDUA 酵素活性降低。含多型性 R105Q、A361T、V454I 的重組質體 ( IDUA/POLYM ) 轉移細胞株所表現的 IDUA 酵素活性、IDUA mRNA、IDUA 蛋白質分別為野生型的 156.9 %、112 %、170 %，顯示多型性 R105Q、A361T、V454I 可促進 IDUA 蛋白質的穩定性，導致 IDUA 酵素活性升高。 在 IDUA 酵素的動力學分析上，分別測定野生型及多型性 IDUA 酵素在不同受質濃度 ( 0.2、1、4 mM ) 下反應 1 小時後生成的產物量，結果顯示多型性的 IDUA 酵素活性約為野生型的 150 %。以 1/V 為 1/[S] 的函數作回歸分析，求得野生型 IDUA 酵素之 Michaelis-Menten 方程式為 Y = 1.1709 X + 3.3007， KM 為 354.7 (M，VMAX 為 2525 pmol/min/mg cell protein；多型性的 IDUA 酵素之 Michaelis-Menten 方程式為 Y = 0.721 X + 2.4145，KM 為 298.6 (M，VMAX 為 3450 pmol/min/mg cell protein。

α-L-Iduronidase ( IDUA ) is one of the lysosomal enzymes involved in the stepwise degradation of glycosaminoglycans heparan sulfate and dermatan sulfate. Loss or marked reduction of IDUA activity results in the autosomal recessive mucopolysaccharidosis type I ( MPS I ) disease. Both IDUA gene and cDNA had been characterized. Mutation screening in patients with MPS I identified a number of mutations and polymorphisms within IDUA gene.In the present study, the effects of previously identified IDUA mutations ( T364M, M1I+Δ54-100, Y343X, and R619G ) and polymorphisms ( R105Q, A361T, and V454I ) on IDUA gene expression, and the kinetic properties of IDUA were investigated. Expression of recombinant IDUA cDNA containing T364M mutation resulted in 1.2 % IDUA activity, 32 % IDUA mRNA but no immunodetectable IDUA protein as compared to those of normal cDNA upon transfection into COS-7 cells. The results revealed that mutation T364M reduced ID activity due to reduction of IDUA mRNA and protein stability. Expression of M1I+Δ54-100, Y343X, R619G resulted in 10.5 %, 4.5 %, 4.9 % IDUA activity and 124 %, 94 %, 80 % IDUA mRNA, respectively, as compared to those of normal cDNA upon transfection into COS-7 cells. The effects of the three mutations were mainly laid on protein level. The M1I+(54-100 protein has in-frame deletion of N-terminal 132 amino acids. Without signal peptide, the mutant protein failed to be transported into lysosome and had low acAtivity as a result. Y343X cDNA coded for IDUA protein with in-frame deletion of 325-343 amino acids including one of the glycosylation sites, Asn-336. No Y343X protein in transfected COS-7 cells was detected. Thus the mutation Y343X may affect the transport and the stability of IDUA protein and result in reduced IDUA activity. About 52 % immunodetectable R619G protein in transfected COS-7 cells was detected as compared to that of wild type. The results suggest that R619G alters the structure of the IDUA protein and results in reduced IDUA activity. Expression of IDUA protein with R105Q, A361T, and V454I polymorphisms resulted in 156.9 % IDUA activity, 112 % IDUA mRNA and 170 % IDUA protein as compared to those of normal cDNA upon transfection into COS-7 cells. The results suggest that the increased activity associating with polymorphic IDUA enzyme is due to enhancing IDUA protein stability.Enzyme kinetic analysis of wild type and polymorphic IDUA was carried out with various concentrations of substrate for one hour reaction. From linear-regression analysis of 1/[S] v.s. 1/V, the Michaelis-Menten equation of wild type IDUA is Y = 1.1709 X + 3.3007, and the KM and VMAX are 354.7 μM and 2525 pmol/min/mg cell protein, respectively. Similarly, the Michaelis-Menten equation of polymorphic IDUA is Y = 0.721 X + 2.4145, and the KM and VMAX are 298.6 μM and 3450 pmol/min/mg cell protein, respectivel