Ishikura Syuhei, Usami Noriyuki, Araki Mayuko, Hara Akira
Laboratory of Biochemistry, Gifu Pharmaceutical University, Mitahora-higashi, Gifu 502-8585.
J Biochem. 2005 Mar;137(3):303-14. doi: 10.1093/jb/mvi033.
L-Gulonate 3-dehydrogenase (GDH) catalyzes the NAD(+)-linked dehydrogenation of L-gulonate into dehydro-L-gulonate in the uronate cycle. In this study, we isolated the enzyme and its cDNA from rabbit liver, and found that the cDNA is identical to that for rabbit lens lambda-crystallin except for lacking a codon for Glu(309). The same cDNA species, but not the lambda-crystallin cDNA with the codon for Glu(309), was detected in the lens, which showed the highest GDH activity among rabbit tissues. In addition, recombinant human lambda-crystallin that lacks Glu(309) displays enzymatic properties similar to rabbit GDH. These data indicate that GDH is recruited as lambda-crystallin without gene duplication. An outstanding feature of GDH is modulation of its activity by low concentrations of P(i), which decreases the catalytic efficiency in a dose dependent manner. P(i) also protects the enzyme against both thermal and urea denaturation. Kinetic analysis suggests that P(i) binds to both the free enzyme and its NAD(H)-complex in the sequential ordered mechanism. Furthermore, we examined the roles of Asp(36), Ser(124), His(145), Glu(157 )and Asn(196) in the catalytic function of rabbit GDH by site-directed mutagenesis. The D36R mutation leads to a switch in favor of NADP(H) specificity, suggesting an important role of Asp(36) in the coenzyme specificity. The S124A mutation decreases the catalytic efficiency 500-fold, and the H145Q, N196Q and N195D mutations result in inactive enzyme forms, although the E157Q mutation produces no large kinetic alteration. Thus, Ser(124), His(145) and Asn(196) may be critical for the catalytic function of GDH.
L-古洛糖酸3-脱氢酶（GDH）在糖醛酸循环中催化L-古洛糖酸与NAD（+）相连的脱氢反应生成脱氢-L-古洛糖酸。在本研究中，我们从兔肝脏中分离出该酶及其cDNA，发现该cDNA与兔晶状体λ-晶体蛋白的cDNA相同，只是缺少编码Glu（309）的密码子。在晶状体中检测到相同的cDNA种类，但未检测到带有编码Glu（309）密码子的λ-晶体蛋白cDNA，晶状体在兔组织中GDH活性最高。此外，缺少Glu（309）的重组人λ-晶体蛋白表现出与兔GDH相似的酶学性质。这些数据表明，GDH被招募为λ-晶体蛋白，无需基因复制。GDH的一个突出特点是低浓度的无机磷酸（P（i））对其活性的调节作用，P（i）以剂量依赖的方式降低催化效率。P（i）还能保护该酶免受热变性和尿素变性。动力学分析表明，P（i）以顺序有序机制与游离酶及其NAD（H）复合物结合。此外，我们通过定点诱变研究了Asp（36）、Ser（124）、His（145）、Glu（157）和Asn（196）在兔GDH催化功能中的作用。D36R突变导致对NADP（H）特异性的偏好转变，表明Asp（36）在辅酶特异性中起重要作用。S124A突变使催化效率降低500倍，H145Q、N196Q和N195D突变导致无活性的酶形式，尽管E157Q突变未产生较大的动力学改变。因此，Ser（124）、His（145）和Asn（196）可能对GDH的催化功能至关重要。
