Johnson W V, Anderson P M
J Biol Chem. 1987 Jul 5;262(19):9021-5.
Cyanase is an inducible enzyme in Escherichia coli that catalyzes bicarbonate-dependent decomposition of cyanate to ammonia and bicarbonate. Previous studies provided evidence that carbamate is an initial product and that the kinetic mechanism is rapid equilibrium random (bicarbonate serving as substrate as opposed to activator); the following mechanism was proposed (Anderson, P. M. (1980) Biochemistry 19, 2282-2888; Anderson, P. M., and Little, R. M. (1986) Biochemistry 25, 1621-1626). (formula; see text) Direct evidence for this mechanism was obtained in this study by 1) determining whether CO2 or HCO3- serves as substrate and is formed as product, 2) identifying the products formed from [14C]HCO3- and [14C] OCN-, 3) identifying the products formed from [13C] HCO3- and [12C]OCN- in the presence of [18O]H2O, and 4) determining whether 18O from [18O]HCO3- is incorporated into CO2 derived from OCN-. Bicarbonate (not CO2) is the substrate. Carbon dioxide (not HCO3-) is produced in stoichiometric amounts from both HCO3- and OCN-. 18O from [18O]H2O is not incorporated into CO2 formed from either HCO3- or OCN-. Oxygen-18 from [18O]HCO3- is incorporated into CO2 derived from OCN-. These results support the above mechanism, indicating that decomposition of cyanate catalyzed by cyanase is not a hydrolysis reaction and that bicarbonate functions as a recycling substrate.
氰酸酶是大肠杆菌中的一种可诱导酶，它催化氰酸盐在碳酸氢盐存在下分解为氨和碳酸氢盐。先前的研究表明氨基甲酸盐是初始产物，且动力学机制为快速平衡随机机制（碳酸氢盐作为底物而非激活剂）；提出了以下机制（安德森，P.M.（1980年）《生物化学》19卷，2282 - 2888页；安德森，P.M.和利特尔，R.M.（1986年）《生物化学》25卷，1621 - 1626页）。（公式；见正文）本研究通过以下方式获得了该机制的直接证据：1）确定CO₂或HCO₃⁻是作为底物并作为产物形成，2）鉴定由[¹⁴C]HCO₃⁻和[¹⁴C]OCN⁻形成的产物，3）在[¹⁸O]H₂O存在下鉴定由[¹³C]HCO₃⁻和[¹²C]OCN⁻形成的产物，以及4）确定来自[¹⁸O]HCO₃⁻的¹⁸O是否掺入由OCN⁻衍生的CO₂中。碳酸氢盐（而非CO₂）是底物。CO₂（而非HCO₃⁻）以化学计量的量由HCO₃⁻和OCN⁻两者产生。来自[¹⁸O]H₂O的¹⁸O未掺入由HCO₃⁻或OCN⁻形成的CO₂中。来自[¹⁸O]HCO₃⁻的氧 - 18掺入由OCN⁻衍生的CO₂中。这些结果支持上述机制，表明氰酸酶催化的氰酸盐分解不是水解反应，且碳酸氢盐起循环底物的作用。
