Orotidine 5′-Monophosphate Decarboxylase: The Operation of Active Site Chains Within and Across Protein Subunits
Orotidine 5′-Monophosphate Decarboxylase: The Operation of Active Site Chains Within and Across Protein Subunits
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DOI：
10.1021/acs.biochem.0c00241
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发表时间：
2020-06-02
期刊：
影响因子：
2.9
通讯作者：
Richard, John P.
中科院分区：
文献类型：
--
作者：
Brandao, Tiago A. S.;Richard, John P.
orotidine 5‘-单磷酸脱羧酶（OMPDC）的D37侧链和T100’侧链分别与结合底物的C-3‘和C-2’核糖基羟基相互作用。通过确定D37G、D37A、T100’g和T100’a取代对以下方面的影响，我们比较了D37的亚基内相互作用与T100’亚基间相互作用：(a) OMPDC催化的OMP和5-氟罗替丁5’-单磷酸（FOMP）脱羧反应的k（cat）和k(cat)/ k -m值，以及(b)二聚体OMPDC相对于单体的稳定性。D37G和t100’a取代导致OMP脱羧k(cat)/ k -m δ G（匕首）增加2 kcal mol(-1)，而D37A和t100’G取代导致δ G（匕首）δ G（匕首）分别增加4和5 kcal mol（-1）。与OMP相比，D37G和T100'A取代均导致FOMP脱羧的δ G（匕首）减少2千卡摩尔（-1）。这些结果表明，D37G和T100'A取代会影响化学脱羧步骤的屏障，而D37A和T100'G取代也会影响配体驱动的缓慢酶构象变化的屏障。底物结合诱导α -螺旋（G'98-S‘106）向底物C-2’核糖基主亚基羟基结合方向移动。与单体相比，T100'G取代使酶二聚体失稳3.5千卡摩尔（-1），这与已知的内部Gly侧链对α -螺旋的失稳一致[Serrano， L., et al. (1992) Nature, 356, 453-455]。我们认为T100'G取代削弱了二聚体界面上的α -螺旋接触，导致二聚体稳定性下降，并增加了配体驱动构象变化的屏障。
The D37 and T100' side chains of orotidine 5'-monophosphate decarboxylase (OMPDC) interact with the C-3' and C-2' ribosyl hydroxyl groups, respectively, of the bound substrate. We compare the intra-subunit interactions of D37 with the inter-subunit interactions of T100' by determining the effects of the D37G, D37A, T100'G, and T100'A substitutions on the following: (a) k(cat) and k(cat)/K-m values for the OMPDC-catalyzed decarboxylations of OMP and 5-fluoroorotidine 5'-monophosphate (FOMP) and (b) the stability of dimeric OMPDC relative to the monomer. The D37G and T100'A substitutions resulted in 2 kcal mol(-1) increases in Delta G(dagger) for k(cat)/K-m, for the decarboxylation of OMP, while the D37A and T100'G substitutions resulted in larger 4 and 5 kcal mol(-1) increases, respectively, in Delta G(dagger). The D37G and T100'A substitutions both resulted in smaller 2 kcal mol(-1) decreases in Delta G(dagger) for the decarboxylation of FOMP compared to that of OMP. These results show that the D37G and T100'A substitutions affect the barrier to the chemical decarboxylation step while the D37A and T100'G substitutions also affect the barrier to a slow, ligand-driven enzyme conformational change. Substrate binding induces the movement of an alpha-helix (G'98-S'106) toward the substrate C-2' ribosyl hydroxy bound at the main subunit. The T100'G substitution destabilizes the enzyme dimer by 3.5 kcal mol(-1) compared to the monomer, which is consistent with the known destabilization of alpha-helices by the internal Gly side chains [Serrano, L., et al. (1992) Nature, 356, 453-455]. We propose that the T100'G substitution weakens the alpha-helical contacts at the dimer interface, which results in a decrease in the dimer stability and an increase in the barrier to the ligand-driven conformational change.