Larimer F W, Harpel M R, Hartman F C
Protein Engineering Program, Oak Ridge National Laboratory, Tennessee 37831-8077.
J Biol Chem. 1994 Apr 15;269(15):11114-20.
Five residues (Thr-53, Asn-54, Gly-370, Gly-393, and Gly-394) of Rhodospirillum rubrum ribulose-bisphosphate carboxylase/oxygenase are positioned to serve as hydrogen-bond donors for the C1 phosphate of ribulose bisphosphate and thereby constrain conformational flexibility of the initial enediol(ate) intermediate (Knight, S., Andersson, I., and Brändén, C.-I. (1990) J. Mol. Biol. 215, 113-160). To study the functional contributions of the residues implicated in ribulose bisphosphate binding and intermediate stabilization, we have replaced them individually with alanine, either to remove the H-bonding group (T53A, N54A) or to introduce bulk (G370A, G393A, G394A). Consequences of substitutions include diminution of carboxylase activity (with a lesser impact on enolization activity), increase of Km (ribulose bisphosphate), and decrease of carboxylation: oxygenation specificity. During catalytic turnover of ribulose bisphosphate by several mutants, substantial amounts of the substrate are diverted to 1-deoxy-D-glycero-2,3-pentodiulose 5-phosphate, reflecting beta-elimination of phosphate from the enediol(ate) intermediate. This side product is not observed with wild-type enzyme, nor has it been reported with mutant enzymes characterized previously. Another consequence of disruption of the phosphate binding site is enhanced production of pyruvate, relative to wild-type enzyme, by some of the mutants due to decomposition of the acicarbanion of 3-phosphoglycerate (the terminal intermediate). These data provide direct evidence that phosphate ligands stabilize conformations of intermediates that favor productive turnover and mitigate beta-elimination at two stages of overall catalysis.
深红红螺菌核酮糖-1,5-二磷酸羧化酶/加氧酶的五个残基（苏氨酸-53、天冬酰胺-54、甘氨酸-370、甘氨酸-393和甘氨酸-394）的位置可作为核酮糖-1,5-二磷酸C1磷酸基团的氢键供体，从而限制初始烯二醇（盐）中间体的构象灵活性（奈特，S.，安德森，I.，和布兰登，C.-I.（1990年）《分子生物学杂志》215卷，113 - 160页）。为了研究与核酮糖-1,5-二磷酸结合及中间体稳定化相关残基的功能贡献，我们将它们逐个替换为丙氨酸，要么去除氢键基团（T53A、N54A），要么引入体积较大的基团（G370A、G393A、G394A）。替换的结果包括羧化酶活性降低（对烯醇化活性影响较小）、Km（核酮糖-1,5-二磷酸）增加以及羧化：加氧特异性降低。在几种突变体催化核酮糖-1,5-二磷酸周转的过程中，大量底物转向1-脱氧-D-甘油-2,3-戊二酮糖5-磷酸，这反映了从烯二醇（盐）中间体上β-消除磷酸基团。野生型酶未观察到这种副产物，之前表征的突变体酶也未报道过。磷酸结合位点破坏的另一个结果是，一些突变体相对于野生型酶，由于3-磷酸甘油酸（末端中间体）的酸式碳酸根分解，丙酮酸产量增加。这些数据提供了直接证据，表明磷酸配体稳定了有利于有效周转的中间体构象，并在整个催化的两个阶段减轻了β-消除。
