Munagala N R, Wang C C
Department of Pharmaceutical Chemistry, School of Pharmacy, University of California, San Francisco 94143-0446, USA.
Biochemistry. 1998 Nov 24;37(47):16612-9. doi: 10.1021/bi9818974.
The hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRTase) from Tritrichomonas foetus has been proven to be a target for potential anti-tritrichomonial chemotherapy. Using a structure-based approach, the base-binding region of the active site of this enzyme, which confers unique purine base specificity, was characterized using site-directed mutagenesis. Determining the roles of different active-site residues in purine specificity would form the basis for designing specific inhibitors toward the parasitic enzyme. A D163N mutant converts the HGXPRTase into a HGPRTase, which no longer recognizes xanthine as a substrate, whereas specificities toward guanine and hypoxanthine are unaffected. Apparently, the side-chain carboxyl of Asp163 forms a hydrogen bond through a water molecule with the C2-carbonyl of xanthine, which constitutes the critical force enabling the enzyme to recognize xanthine as a substrate. Mutations of Arg155, which orients and stacks the neighboring Tyr156 onto the bound purine base by forming a salt bridge between itself and Glu11, result in drastic increases in the Kms for GMP and XMP (but not IMP). This change leads to increased kcats for the forward reactions with guanine and xanthine as substrates without affecting the conversion of hypoxanthine to IMP. Thus, the apparent dislocation of Tyr156, resulted from mutations of Arg155, bring little effect on the hydrophobic interactions between Tyr156 and the purine ring. But the forces involved in recognizing the exocyclic C2-substituents of the purine ring, which involve the Tyr156 hydroxyl, Ile157 backbone carbonyl, and Asp163 side-chain carboxyl, may be weakened by the shifted conformation of the peptide backbone resulted from loss of the Glu11-Arg155 salt bridge. The conserved Lys134 was proven to be the primary determinant in conferring the specificity of the enzyme toward 6-oxopurines. By substituting the lysine residue for a serine, which can potentially hydrogen bond to either an amino or an oxo-group, we have successfully augmented the purine specificity of the enzyme. The K134S mutant recognizes adenine in addition to hypoxanthine, guanine, and xanthine as its substrates. Adenine and hypoxanthine are equivalent substrates for the mutant enzyme with similar Kms of 34.6 and 38.0 microM, respectively. The catalysis of an adenine phosphoribosyltransferase reaction by this mutant enzyme was further demonstrated by the competitive inhibition of AMP with an estimated Kis of 25.4 microM against alpha-D-5-phosphoribosyl-pyrophosphate (PRPP) in converting hypoxanthine to IMP. We have thus succeeded in using site-directed mutagenesis to convert T. foetusHGXPRTase into either a HGPRTase or a genuine AHGXPRTase.
已证实胎儿三毛滴虫的次黄嘌呤 - 鸟嘌呤 - 黄嘌呤磷酸核糖转移酶（HGXPRTase）是潜在抗三毛滴虫化疗的一个靶点。采用基于结构的方法，利用定点诱变对该酶活性位点的碱基结合区域进行了表征，该区域赋予了独特的嘌呤碱基特异性。确定不同活性位点残基在嘌呤特异性中的作用将为设计针对这种寄生酶的特异性抑制剂奠定基础。D163N突变体将HGXPRTase转变为HGPRTase，后者不再将黄嘌呤识别为底物，而对鸟嘌呤和次黄嘌呤的特异性不受影响。显然，Asp163的侧链羧基通过一个水分子与黄嘌呤的C2 - 羰基形成氢键，这构成了使该酶将黄嘌呤识别为底物的关键作用力。Arg155通过与Glu11形成盐桥来定向并将相邻的Tyr156堆叠在结合的嘌呤碱基上，其突变导致GMP和XMP（但不包括IMP）的米氏常数（Km）大幅增加。这种变化导致以鸟嘌呤和黄嘌呤为底物的正向反应的催化常数（kcats）增加，而不影响次黄嘌呤向IMP的转化。因此，由Arg155突变导致的Tyr156明显错位对Tyr156与嘌呤环之间的疏水相互作用影响不大。但是，涉及Tyr156羟基、Ile157主链羰基和Asp163侧链羧基的识别嘌呤环外环C2 - 取代基的作用力可能会因Glu11 - Arg155盐桥的丧失导致的肽主链构象改变而减弱。已证明保守的Lys134是赋予该酶对6 - 氧嘌呤特异性的主要决定因素。通过用丝氨酸取代赖氨酸残基，丝氨酸可能与氨基或氧代基团形成氢键，我们成功增强了该酶的嘌呤特异性。K134S突变体除了将次黄嘌呤、鸟嘌呤和黄嘌呤识别为底物外，还能识别腺嘌呤。腺嘌呤和次黄嘌呤是该突变酶的等效底物，其米氏常数分别为34.6和38.0 microM，相似。该突变酶催化腺嘌呤磷酸核糖转移酶反应进一步通过在将次黄嘌呤转化为IMP过程中AMP对α - D - 5 - 磷酸核糖 - 焦磷酸（PRPP）的竞争性抑制得到证明，估计抑制常数（Kis）为25.4 microM。因此，我们成功地利用定点诱变将胎儿三毛滴虫HGXPRTase转变为HGPRTase或真正的AHGXPRTase。
