Lundegaard C, Jensen K F
Center for Enzyme Research, Institute of Molecular Biology, University of Copenhagen, Denmark.
Biochemistry. 1999 Mar 16;38(11):3327-34. doi: 10.1021/bi982279q.
Phosphoribosyltransferases catalyze the formation of nucleotides from a nitrogenous base and 5-phosphoribosyl-alpha-1-pyrophosphate (PRPP). These enzymes and the PRPP synthases resemble each other in a short homologous sequence of 13 amino acid residues which has been termed the PRPP binding site and which interacts with the ribose 5-phosphate moiety in structurally characterized complexes of PRPP and nucleotides. We show that each class of phosphoribosyltransferases has subtle deviations from the general consensus PRPP binding site and that all uracil phosphoribosyltransferases (UPRTases) have a proline residue at a position where other phosphoribosyltransferases and the PRPP synthases have aspartate. To investigate the role of this unusual proline (Pro 131 in the E. coli UPRTase) for enzyme activity, we changed the residue to an aspartate and purified the mutant P131D enzyme to compare its catalytic properties with the properties of the wild-type protein. We found that UPRTase of E. coli obeyed the kinetics of a sequential mechanism with the binding of PRPP preceding the binding of uracil. The basic kinetic constants were derived from initial velocity measurements, product inhibition, and ligand binding assays. The change of Pro 131 to Asp caused a 50-60-fold reduction of the catalytic rate (kcat) in both directions of the reaction and approximately a 100-fold increase in the KM for uracil. The KM for PRPP was strongly diminished by the mutation, but kcat/KM,PRPP and the dissociation constant (KD,PRPP) were nearly unaffected. We conclude that the proline in the PRPP binding site of UPRTase is of only little importance for binding of PRPP to the free enzyme, but is critical for binding of uracil to the enzyme-PRPP complex and for the catalytic rate.
磷酸核糖转移酶催化由含氮碱基和5-磷酸核糖-α-1-焦磷酸（PRPP）形成核苷酸。这些酶和PRPP合酶在一段由13个氨基酸残基组成的短同源序列中彼此相似，该序列被称为PRPP结合位点，并且在PRPP与核苷酸的结构特征复合物中与5-磷酸核糖部分相互作用。我们发现，每一类磷酸核糖转移酶与一般的PRPP结合位点共识都有细微偏差，并且所有尿嘧啶磷酸核糖转移酶（UPRTase）在其他磷酸核糖转移酶和PRPP合酶具有天冬氨酸的位置上都有一个脯氨酸残基。为了研究这个不寻常的脯氨酸（大肠杆菌UPRTase中的Pro 131）对酶活性的作用，我们将该残基替换成天冬氨酸，并纯化了突变型P131D酶，以将其催化特性与野生型蛋白的特性进行比较。我们发现大肠杆菌的UPRTase遵循有序机制的动力学，PRPP的结合先于尿嘧啶的结合。基本动力学常数来自初始速度测量、产物抑制和配体结合测定。将Pro 131替换为Asp导致反应两个方向的催化速率（kcat）降低50 - 60倍，并且尿嘧啶的KM增加约100倍。突变使PRPP的KM大幅降低，但kcat/KM,PRPP和解离常数（KD,PRPP）几乎不受影响。我们得出结论，UPRTase的PRPP结合位点中的脯氨酸对PRPP与游离酶的结合重要性不大，但对尿嘧啶与酶 - PRPP复合物的结合以及催化速率至关重要。
