Abend A, Garrison P N, Barnes L D, Frey P A
Institute for Enzyme Research, The Graduate School, and Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin-Madison, Madison, Wisconsin 53705, USA.
Biochemistry. 1999 Mar 23;38(12):3668-76. doi: 10.1021/bi981895j.
Fhit is the protein product of FHIT, a candidate human tumor suppressor gene. Fhit catalyzes the hydrolysis of diadenosine triphosphate (Ap3A) to AMP and ADP. Fhit is here shown to catalyze the hydrolysis in H218O with production of adenosine 5'-[18O]phosphate and ADP, proving that the substitution of water is at Palpha and not at Pbeta. The chain fold of Fhit is similar to that of galactose-1-phosphate uridylyltransferase, which functions by a double-displacement mechanism through the formation of a covalent nucleotidyl-enzyme intermediate and overall retention of configuration at Palpha. The active site of Fhit contains a histidine motif that is reminiscent of the HPH motif in galactose-1-phosphate uridylyltransferases, in which the first histidine residue serves as the nucleophilic catalyst to which the nucleotidyl group is bonded covalently in the covalent intermediate. In this work, the Fhit-catalyzed cleavage of (RP)- and (SP)-gamma-(m-nitrobenzyl) adenosine 5'-O-1-thiotriphosphate (mNBATPalphaS) in H218O to adenosine 5'-[18O]thiophosphate is shown to proceed with overall retention of configuration at phosphorus. gamma-(m-Nitrobenzyl) adenosine 5'-O-triphosphate (mNBATP) is approximately as good a substrate for Fhit as Ap3A, and both (RP)- and (SP)-mNBATPalphaS are substrates that react at about 0.5% of the rate of Ap3A. The stereochemical evidence indicates that hydrolysis by Fhit proceeds by a double-displacement mechanism, presumably through a covalent AMP-enzyme intermediate.
Fhit是候选人类肿瘤抑制基因FHIT的蛋白质产物。Fhit催化三磷酸二腺苷（Ap3A）水解为AMP和ADP。本文显示Fhit在H₂¹⁸O中催化水解，生成5'-[¹⁸O]磷酸腺苷和ADP，证明水的取代发生在α位磷原子而非β位磷原子。Fhit的链折叠与1-磷酸半乳糖尿苷酰转移酶相似，后者通过形成共价核苷酸-酶中间体的双取代机制发挥作用，且在α位磷原子处构型总体保持不变。Fhit的活性位点包含一个组氨酸基序，这让人联想到1-磷酸半乳糖尿苷酰转移酶中的HPH基序，其中第一个组氨酸残基作为亲核催化剂，在共价中间体中核苷酸基团与之共价结合。在这项工作中，Fhit催化（RP）-和（SP）-γ-（间硝基苄基）腺苷5'-O-1-硫代三磷酸（mNBATPαS）在H₂¹⁸O中裂解为5'-[¹⁸O]硫代磷酸腺苷，结果表明在磷原子处构型总体保持不变。γ-（间硝基苄基）腺苷5'-O-三磷酸（mNBATP）作为Fhit的底物与Ap3A的效果大致相同，并且（RP）-和（SP）-mNBATPαS都是底物，其反应速率约为Ap3A的0.5%。立体化学证据表明Fhit催化的水解通过双取代机制进行，大概是通过共价AMP-酶中间体。
