Munagala N R, Chin M S, Wang C C
Department of Pharmaceutical Chemistry, School of Pharmacy, University of California, San Francisco 94143-0446, USA.
Biochemistry. 1998 Mar 24;37(12):4045-51. doi: 10.1021/bi972515h.
Tritrichomonas foetus, an anaerobic flagellated protozoan, causes urogenital trichomoniasis in cattle. Hypoxanthine-guanine-xanthine phosphoribosyl transferase (HGXPRTase), an essential enzyme in T. foetus required for salvaging exogenous purine bases, has been regarded as a promising target for anti-tritrichomonial chemotherapy. The steady-state kinetic analyses of synthesis and pyrophosphorolysis of IMP, GMP, and XMP and product inhibition studies have been used to elucidate the reaction mechanisms. Double-reciprocal plots of initial velocities versus the varying concentrations of one substrate at a fixed concentration of the other show intersecting lines indicating a sequential mechanism for both the forward and the reverse reactions. In terms of the kcat/Km ratios, hypoxanthine is the most effective substrate whereas guanine and xanthine are converted equally well into their corresponding nucleotides. The minimum kinetic model from the data in product inhibition studies is an ordered bi-bi mechanism, where the substrates bind to the enzyme (first PRPP followed by the purine bases), and the products released (first PPi followed by purine nucleotide) in a defined order. The Kms for PPi in the T. foetus HGXPRTase-catalyzed reactions are unusually high, close to the millimolar range. Since the crystal structure of this enzyme [Somoza et al. (1996) Biochemistry 35, 7032-7040] suggests potential binding between the threonine-47 in a conserved cis-peptide loop and PPi whereas human HGPRTase has lysine-68 [Eads et al. (1994) Cell 78, 325-334] at the corresponding position, we prepared a T47K enzyme mutant and found in the T47K-catalyzed reaction a 4-10-fold decrease of Km for PPi. The lack of ionic interactions between Thr-47 and PPi and an increased distance between the loop and the active site as compared to the human HGPRTase are thus proposed to be responsible for the high Km for PPi in the T. foetus HGXPRTase-catalyzed reaction.
胎儿三毛滴虫是一种厌氧的有鞭毛原生动物，可引起牛的泌尿生殖系统滴虫病。次黄嘌呤 - 鸟嘌呤 - 黄嘌呤磷酸核糖转移酶（HGXPRTase）是胎儿三毛滴虫中挽救外源性嘌呤碱基所需的一种必需酶，被认为是抗三毛滴虫化疗的一个有前景的靶点。通过对IMP、GMP和XMP的合成及焦磷酸解的稳态动力学分析以及产物抑制研究来阐明反应机制。在固定另一种底物浓度的情况下，初始速度与一种底物不同浓度的双倒数图显示相交线，表明正向和反向反应均为顺序机制。就kcat/Km比值而言，次黄嘌呤是最有效的底物，而鸟嘌呤和黄嘌呤转化为其相应核苷酸的效果相当。产物抑制研究数据得出的最小动力学模型是有序的双底物双产物机制，即底物按一定顺序与酶结合（先结合PRPP，再结合嘌呤碱基），产物按一定顺序释放（先释放PPi，再释放嘌呤核苷酸）。在胎儿三毛滴虫HGXPRTase催化的反应中，PPi的Km值异常高，接近毫摩尔范围。由于该酶的晶体结构[索莫扎等人（1996年）《生物化学》35卷，7032 - 7040页]表明在保守的顺式肽环中的苏氨酸 - 47与PPi之间可能存在结合，而人HGPRTase在相应位置有赖氨酸 - 68[伊兹等人（1994年）《细胞》78卷，325 - 334页]，我们制备了T47K酶突变体，发现在T47K催化的反应中，PPi的Km值降低了4 - 10倍。因此，有人提出，与人类HGPRTase相比，Thr - 47与PPi之间缺乏离子相互作用以及环与活性位点之间距离增加，是胎儿三毛滴虫HGXPRTase催化反应中PPi的Km值高的原因。
