Buffer enhancement of proton transfer in catalysis by human carbonic anhydrase III.
Buffer enhancement of proton transfer in catalysis by human carbonic anhydrase III.
复制标题
人碳酸酐酶 III 催化中质子转移的缓冲增强。
DOI：
10.1021/bi00479a009
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发表时间：
1990
期刊：
影响因子：
2.9
通讯作者：
Silverman,DN
中科院分区：
文献类型：
--
作者：
Tu,CK;Paranawithana,SR;Jewell,DA;Tanhauser,SM;LoGrasso,PV;Wynns,GC;Laipis,PJ;Silverman,DN
材料与方法酶。含有野生型HCA III基因的细菌表达载体来源于Lloyd等人的cDNA克隆。（1986）使用pET-8载体（F. William Studier，布鲁克海文国家实验室）。这类表达载体由Rosenberg et al.（1987年）。按Kunkel（1985）的方法构建了位点特异突变体K64 H、R67 H、R67 N和双突变体K64 H-R67 N，并将其转入同一pET-8载体中。在大肠杆菌菌株BL 21（DE 3）pLysS中表达修饰的和未修饰的碳酸酐酶III（Rosenberg等人，1987），并通过凝胶过滤，然后如Tu et al.（1986年）。用聚丙烯酰胺凝胶电泳测定，所得酶的纯度大于98%。野生型HCA III和突变体的浓度由在280 nm处的摩尔吸光系数6.2 X 104 M™ 1 cm-1确定（Sanyal等人，1982），与牛同工酶III发现的值相同（Engberg等，1985年）。180交换动力学。膜入口质谱法用于测量在水合-脱水循环期间发生的CO2和水之间的未催化的和碳酸酐酶催化的180交换。还测量了12 C-和13 C-标记的CO2之间的180交换;这是因为催化的脱水导致活性位点被180短暂标记，然后与13 CO2反应（Silverman等人，1979; Silverman，1982）。从180交换速率可以得到两个化学平衡下的催化速率.第一个速率Ri是CO2和HCO 3 '相互转化的催化速率，碳酸酐酶II的速率与13 C NMR获得的速率一致（Silverman等人，1979年）。第二种，7？H2O是180标记的水从酶释放的速率。用180瞬时标记活性位点还提供了180标记的水从酶中释放的速率的测量，这是一个质子转移依赖性过程，因为预期重新标记的氢氧化物不会快速交换（等式3; Silverman等人，1979; Silverman & Lindskog，1988）。这里
Materials and MethodsEnzymes. A bacterial expression vector containing a wild-type HCA III gene was derived from the cDNA clone of Lloyd et al.(1986) using the pET-8 vector (a gift from Dr. F. William Studier, Brookhaven National Laboratory). This class of expression vectors is described by Rosenberg et al.(1987). The site-specific mutants K64H, R67H, and R67N and the double mutant K64H-R67N were constructed ac-cording to the method of Kunkel (1985) and transferred into the same pET-8 vector. Modified and unmodified carbonic anhydrases III were expressed in Escherichia coli strain BL21 (DE3) pLysS (Rosenberg et al., 1987) and purified by gel filtration followed by ion-exchange chromatography as described by Tu et al.(1986). Theresulting enzymes were greater than 98% pure, determined by polyacrylamide gel electrophoresis. The concentrations of wild-type HCA III and mutants were determined from the molar absorptivity of 6.2 X 104 M™ 1 cm'1 at 280 nm (Sanyal et al., 1982), identical with the value found for bovine isozyme III (Engberg et al., 1985). 180 Exchange Kinetics. Membrane-inlet mass spectrometry was used to measure the uncatalyzed and carbonic anhydrase catalyzed exchange of l80 between C02 and water which occurs during the hydration-dehydration cycle. The exchange of 180 between 12C-and 13C-labeled C02 was also measured; this occurs because the catalyzed dehydration results in a transitory labeling of the active site with 180 which then reacts with 13C02 (Silverman et al., 1979; Silverman, 1982). Two rates for the catalysisat chemical equilibrium can be obtained from these rates of 180 exchange. The first rate, R {, is the catalyzed rate of interconversion of C02 and HC03', a rate which for carbonic anhydrase II agrees with that obtainedby l3C NMR (Silverman et al., 1979). The second rate, 7? h2o< is the rate of release from the enzyme of 180-labeled water. The transient labeling of the active site with l80 also provides this measure of the rate of release of l80-labeled water from the enzyme, a proton transfer dependentprocess since re-labeled hydroxide is not expected to exchange rapidly (eq 3; Silverman et al., 1979; Silverman & Lindskog, 1988). Here