Volume 62, number 1 FEBS LETTERS February 1976 THE CATALYTIC MECHANISM OF HUMAN CARBONIC ANHYDRASE C: INHIBITION OF CO2 HYDRATION AND ESTER HYDROLYSIS BY HCO~ H. STEINER, B.-H. JONSSON and S. LINDSKOG Institutionen f6r biokemi, G6teborgs Universitet och Chalmers Tekniska H6gskola, Fack, S-402 20 G6teborg 5, Sweden Received 12 November 1975 1. Introduction The transport of I-1 + between the active site and the solvent is a crucial step in the carbonic anhydrase- catalyzed interconversion of CO2 and HCO~ [1,2]. Recently we reported that the steady-state parameters for both directions of the reaction are subject to sub- stantial hydrogen isotope effects, and we put forward the hypothesis that the rate of catalysis is limited by the transfer of H+between a 'catalytic group' and a 'proton transfer group' [2]. The latter group was assumed to exchange H + rapidly with a buffered medium. The results were most easily rationalized by a reaction scheme of the following form: kl IS] k2 HE "~ HX "" HEH + x \ 1L 1L E- " X- x EH k_~ ~-~ [P-I In this scheme the protonated forms of the catalytic group and the proton transfer group are indicated by EH and HE, respectively, while S and P-represent CO2 and HCO3, respectively. The transitory complexes, E--S and EH-P-, are symbolized by X_ The intramolecular proton transfer step, EH , HE, represents an isomerization of stable enzyme forms occurring between the release of product and the binding of substrate. Thus, it should be possible to test our hypothesis by studies of product inhibition [3]. In this paper we report data on the inhibition of CO2 hydration by HCOg. We have also estimated sub- strate binding to carbonic anhydrase C from the inhibition of the esterase activity by equilibrium mix- tures of CO2 and HCOg. The results of these experi- ments are in accordance with the proposed mechanism and suggest that the intramolecular proton transfer step is not completely rate limiting in 1H20 but becomes rate limiting when the solvent is changed to 2H20. 2. Materials and methods Human carbonic anhydrase C was prepared by the E- "~ HE k-3 KE2 (1) method of Henderson and Henriksson [4]. Enzyme concentrations were estimated spectrophotometri- cally at 280 nm taking al% = 18.7 cm -1 [5] and a • " 280 mol. wt. of 29 300 [6]. 2(N-morpholino)ethane- sulfonic acid (MES) and N-2-hydroxethylpiperazine- ~-ethanesulfonic acid (HEPES) were obtained from Sigma Chemical Co. Other chemicals were the same as used in previous investigations [2,7]. Stock solu- tions of CO2 and NaHCOa were prepared as described previously [2]. The CO2 hydration reaction was 16 North-Holland Publishing Company - Amsterdam