Catalysis in human hypoxanthine-guanine phosphoribosyltransferase: Asp 137 acts as a general acid/base.
Catalysis in human hypoxanthine-guanine phosphoribosyltransferase: Asp 137 acts as a general acid/base.
复制标题
人次黄嘌呤鸟嘌呤磷酸核糖基转移酶的催化作用：Asp 137 充当一般酸/碱。
DOI：
10.1021/bi972519m
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发表时间：
1998
期刊：
影响因子：
--
通讯作者：
Grubmeyer,C
中科院分区：
文献类型：
--
作者：
Xu,Y;Grubmeyer,C
次黄嘌呤-鸟嘌呤磷酸核糖基转移酶（HGPRTase）催化IMP和GMP从其各自的碱基次黄嘌呤（Hx）和鸟嘌呤（Gua）以及磷酸核糖基供体5-磷酸核糖基-1-焦磷酸（PRPP）可逆形成。核苷键的净形成和裂解需要在嘌呤部分去除/添加质子，从而允许酶催化降低与反应相关的能垒。IMP焦磷酸解的kcat的pH曲线显示了pKa为7.9的必需酸性基团，而IMP或GMP形成的kcat的pH曲线显示了必需碱性基团的参与。基于人HGPRT酶的晶体结构，质子化/去质子化可能发生在嘌呤环的N7处，并且Lys 165或Asp 137各自是一般碱/酸的候选者。我们已经构建，纯化和动力学特性的突变HGPRTases来测试这一假设。D137 N与Hx反应生成核苷酸的能力降低18倍，与Gua反应生成核苷酸的能力降低275倍，与IMP焦磷酸解反应生成核苷酸的能力降低500倍。D137 N对核苷酸和PRPP的KD值也较低。D137 N的kcat的pH曲线严重改变。与D137 N相比，K165 Q的kcat在正向反应中仅降低2倍，在反向反应中略有增加。KmandKD值表明K165 Q与底物的相互作用比野生型酶弱。用K165 Q进行的预稳态实验表明，磷酸核糖基转移步骤在正向反应中是快速的，如用野生型所观察到的。相比之下，D137 N表现出较慢的磷酸核糖转移化学，尽管鸟嘌呤（减少3000倍）比次黄嘌呤（减少32倍）受到的影响更大。总之，Asp 137作为HGPRT酶的一般催化酸/碱，Lys 165与底物进行基态相互作用。
Hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) catalyzes the reversible formation of IMP and GMP from their respective bases hypoxanthine (Hx) and guanine (Gua) and the phosphoribosyl donor 5-phosphoribosyl-1-pyrophosphate (PRPP). The net formation and cleavage of the nucleosidic bond requires removal/addition of a proton at the purine moiety, allowing enzymic catalysis to reduce the energy barrier associated with the reaction. The pH profile ofkcatfor IMP pyrophosphorolysis revealed an essential acidic group with pKaof 7.9 whereas those for IMP or GMP formation indicated involvement of essential basic groups. Based on the crystal structure of human HGPRTase, protonation/deprotonation is likely to occur at N7 of the purine ring, and Lys 165 or Asp 137 are each candidates for the general base/acid. We have constructed, purified, and kinetically characterized two mutant HGPRTases to test this hypothesis. D137N displayed an 18-fold decrease inkcatfor nucleotide formation with Hx as substrate, a 275-fold decrease inkcatwith Gua, and a 500-fold decrease inkcatfor IMP pyrophosphorolysis. D137N also showed lowerKDvalues for nucleotides and PRPP. The pH profiles ofkcatfor D137N were severely altered. In contrast to D137N, thekcatfor K165Q was decreased only 2-fold in the forward reaction and was slightly increased in the reverse reaction. TheKmandKDvalues showed that K165Q interacts with substrates more weakly than does the wild-type enzyme. Pre-steady-state experiments with K165Q indicated that the phosphoribosyl transfer step was fast in the forward reaction, as observed with the wild type. In contrast, D137N showed slower phosphoribosyl transfer chemistry, although guanine (3000-fold reduction) was affected much more than hypoxanthine (32-fold reduction). In conclusion, Asp137 acts as a general catalytic acid/base for HGPRTase and Lys165 makes ground-state interactions with substrates.