Importance of the conserved active-site residues Tyr7, Glu106 and Thrl99 for the catalytic function of human carbonic anhydrase I1

Transcription

1 Eur. J. Biochem. 211, (1993) 0 FEBS 1993 Importance of the conserved active-site residues Tyr7, Glu106 and Thrl99 for the catalytic function of human carbonic anhydrase I1 Zhiwei LIANG, Yafeng XUE, Gity BEHRAVAN, Bengt-Harald JONSSON and Sven LINDSKOG Avdelningen for biokemi, Umei Universitet, Sweden (Received October 7, 1992) - EJB The catalytic mechanism of carbonic anhydrase includes the reaction of a zinc-bound hydroxide ion with the CO, substrate. This hydroxide ion is part of a hydrogen-bonded network involving the conserved amino acid residues Thr199, Glu106 and Tyr7. To investigate the functional importance of these residues, a number of site-specific mutants have been made. Thus, Thr199 has been changed to Ala, Glu106 to Ala, Gln and Asp, and Tyr7 to Phe. The effects of these mutations on catalyzed CO, hydration and ester hydrolysis have been measured, as well as the binding of some inhibitors. The results show that the CO, hydration activity of the mutant with Phe7 is only marginally reduced, whereas the esterase activity is larger than that of unmodified enzyme. It is concluded that Tyr7 is not a functionally required element of the hydrogen-bonded network. The CO, hydration activity (kcat as well as kcjkm) and the esterase activity of the mutant with Ala199 are reduced about 100- fold. The affinity for the sulfonamide inhibitor, dansylamide, is only slightly reduced while the mutant has an enhanced affinity for bicarbonate and the anionic inhibitor, SCN-. The activities of the mutants with Ala106 and Gln106 are also reduced. The reduction of the esterase activity is about 100-fold, while k,,, for CO, hydration has decreased by a factor of 1OOO. The parameter kcat/ K, is only about one order of magnitude smaller for these mutants than for the unmodified enzyme. The binding of dansylamide and another sulfonamide inhibitor, acetazolamide, are about 20-times weaker to the mutant with Gln106 than to unmodified enzyme. These results suggest important roles for Thr199 and Glu106 in carbonic anhydrase catalysis. The mutant with Asp106 is almost fully active suggesting that the structure has undergone a compensatory change to maintain the interaction between residue 106 and Thr199. It has long been known that the zinc ion in the active site of carbonic anhydrase is required for catalysis of the reversible hydration of CO, [l, 21 as well as for catalysis of ester hydrolysis [2, 31. In addition to the three histidine residues, 94, 96 and 119, which chelate the zinc ion in a very stable complex, the active site contains a number of conserved amino acid residues, which are connected to the metal-ion center through networks of hydrogen bonds [4]. One of these networks is shown schematically in Fig. 1. It includes the fourth ligand of the tetrahedrally coordinated zinc ion, a water molecule or hydroxide ion which has been assumed to be directly involved in the catalytic reaction as indicated in Eqn (1) [5]. The zinc-bound H,O/OH- is hydrogen-bonded to Thr199, which is connected to a buried glutamic acid residue, Glu106. Glu106 is linked to Tyr7 through a bridging water molecule : E-ZnZ'-OH- + CO, + E-Zn" -HCO; E-ZnZ'-H,O + HCO;. (1) To investigate whether or not these conserved residues are functionally important, mutants of human carbonic anhydrase I1 have been prepared. Thus, Thr199 has been replaced Correspondence to S. Lindskog, Avdelningen for biokemi, Umei Universitet, S Umei, Sweden Enzyme. Carbonic anhydrase (EC ). H20 by Ala, Glu106 by Ala, Gln and Asp, and Tyr7 by Phe. In this paper, we report the functional consequences of these alterations for CO, hydration, ester hydrolysis and inhibitor binding, while the structures of the variants with Ala199, Gln106, Ala106 and Asp106 will be described in a separate paper (Y. Xue, A. Liljas, B.-H. Jonsson and S. Lindskog, unpublished results). A preliminary report of some of the results described in the present paper has been published previously [7]. MATERIALS AND METHODS Enzyme The numbering system of human carbonic anhydrase I is used throughout this paper. In this system, residues 7, 106 and 199 correspond to residues 6, 105 and 197 in the numbering system of isoenzyme IT [S]. In vitro site-directed mutagenesis was first performed in an Amersham system, which is based on the method of Taylor et al. [9], and mutants with Ala199 and Gln106 were then expressed in the lon- Escherichia coli strain SG20043 from a plasmid described previously [lo]. Later, all mutants were made with the Muta-Gene system (Bio-Rad), which is based on the method of Kunkel [ll], and expressed in E. coli strain BL21DE3 from the plasmid paca constructed in the

2 822 Fig. 1. Schematic drawing of the hydrogen-bonding network in the active site of human carbonic anhydrase I1 involving Thr199, Glu106 and Tyr7. From Eriksson et al. [4] Reprinted by permission of Wiley-Liss, a division of John Wiley and Sons, Inc. laboratory of C. Fierke at Duke University [12]. Mutants were verified by dideoxy sequencing [13]. The cell-growth conditions described by Forsman et al. [lo] were used, except that the mutants with Ala199 and Gln106 were induced at room temperature. Unmodified, cloned human carbonic anhydrase I1 and the variants with Ala106, Asp106 and Phe7 were purified by affinity chromatography essentially according to Khalifah et al. [14]. Two methods were used to purify the variants with Ala199 and Gln106. The first method (Y.Xue and B.- H. Jonsson, unpublished data) involves chromatography on the anion exchanger S-Sepharose Fast Flow (Pharmacia) in 20 mm Mes/NaOH, ph 6.2, step-wise eluted with (NH,),SO, followed by hydrophobic-interaction chromatography on phenyl-sepharose in 20 mm MesNaOH, ph 6.2, and 2.0 M (NH,),SO,. This column was eluted by step-wise decreasing the (NH,),SO, concentration. The final purification was achieved by gel filtration on a S-100 HR Sephacryl column in 20 mm Hepes, ph 8.2. It was later found that these mutants have a sufficient affinity for sulfonamide inhibitors to allow purification by a modified affinity-chromatography procedure. The mutant enzymes were found to bind to the resin [14] in 5-10 mm Tris/H,SO, or Ches/NaOH, ph 9. The column was washed with the same buffer and elution was performed with 0.4 M NaN, in 100 mm Tris/H,SO,, ph 7.5. Enzyme concentrations were estimated spectrophotometrically at 280 nm using = 54.7 mm-'. cm-' [15] based on a molecular mass of 29.3 kda [16]. However, an of 53.2 mm-'. cm-' was assumed for the variant with Phe7. Proper folding of purified mutants was checked by recording circular-dichroism spectra at nm on a Jasco-600 spectropolarimeter. Furthermore, the mutants with Ala199, Gln106, Ala106 and Asp106 have been crystallized (Y. Xue, A. Liljas, B.-H. Jonsson and S. Lindskog, unpublished results). Kinetic measurements Initial rates of CO, hydration were measured in a Hi- Tech stopped-flow apparatus at 25 C by the changing phindicator method [ 17, 181. Buffer-indicator pairs were 1,2- dimethylimidazole/h,so, or Taps/NaOH with metacresol purple monitored at 578 nm, imidazole/h,so, or Mops/ NaOH with 4-nitrophenol monitored at 400nm and Mes/ NaOH with chlorophenol red monitored at 574 nm. The ionic strength was usually maintained at 0.1 M with Na,SO,. Mes buffers were treated with Chelex 100 resin, and 0.1 mm EDTA was usually included in other buffers to avoid adventitious metal ions. However, 0.1 mm EDTA was found partially to remove the zinc ion from the mutants with Ala199, Ala106 and Gln106, so the chelator was excluded in these cases. In addition, it was later found that sulfate inhibits the mutants with Ala106 and Gln106 and experiments were, therefore, also performed in the absence of this anion. Rate data were fitted to the Michaelis-Menten equation using the program GraFit (Erithacus Software Ltd, UK). Initial rates of 4-nitrophenyl acetate hydrolysis were measured spectrophotometrically at 348 nm and 25 "C using a substrate concentration of 0.4 mm, which is well below K,,, for unmodified enzyme [2]. The apparent second-order rate constants, k,, (kcajkm), were calculated from the total initial rates after subtraction of the non-enzymic rates using = 5.15 mm-'. cm-' [2]. Inhibitor binding A Shimadzu RF-500 spectrofluorimeter was used to study inhibitor binding. Dissociation constants for dansylamide (5-dimethylaminonaphthalene-1-sulfonamide) were estimated from the increase of fluorescence intensity (excitation at 320 nm, emission spectrum recorded at nm) associated with the binding of the inhibitor to the enzyme [19]. Experiments were performed either at a fixed enzyme concentration and varying the dansylamide concentration or at a fixed dansylamide concentration and varying the enzyme concentration. Dissociation constants for other inhibitors were estimated in competition experiments at fixed concentrations of enzyme and dansylamide following the decrease of fluorescence intensity as the concentration of the investigated inhibitor was increased. RESULTS CO, hydration The replacement of Thr199 with Ala in human carbonic anhydrase I1 has a drastic effect on the CO, hydration activity but does not result in a completely inactive enzyme. The observed rates of catalysis follow Michaelis-Menten patterns. The results of measurements in a number of buffers at various ph values are shown in Table 1. While K, for the mutant is approximately independent of ph and has a magnitude similar to the ph-independent K,,, for the unmodified enzyme, the maximal value of k,,, at alkaline ph is only about 1% of that for native human carbonic anhydrase 11. Control experiments, performed in the absence of added Na,SO,, ph 7.2, showed that this mutant is not significantly inhibited by the concentration of sulfate normally used for control of ionic strength (33 mm). The ph dependence of the kinetic parameter, k,,,/k,, is shown graphically in Fig. 2. The data suggest an activity-linked pk, near 8, which is about 1 higher

3 823 Table 1. Kinetic parameters for CO, hydration catalyzed by human Thr Ala carbonic anhydrase 11. Temperature, 25 "C. DMI, 1,2-dimethylimidazole. Buffer concentration, 50 mm. The ionic strength was kept at 0.1 M by addition of NgSO,. Parameter values and standard deviations were obtained by non-linear-regression Buffer PH k,, Km kc.jkm ms-' mm FM-'. s-1 Taps t 0.1 DMI t 0.1 DMI t 0.03 Imidazole ? t 0.01 Mes If: Mes t Fig. 2. ph dependence of k,.jk,,, for CO, hydration catalyzed by human Thr Ala carbonic anhydrase 11. Temperature, 25 C; ionic strength, 0.1 M. The curve has been drawn as a simple titration curve with pk, = 8.1 than the corresponding pk, for unmodified enzyme (cf. Fig. 3). This shows that the activity measured for the mutant is a genuine property of the mutant and not due to the presence of a small quantity of unmodified enzyme in the preparation. The importance of Glu106 was investigated with three mutants having Gln, Ala or Asp at sequence position 106. The variant with Gln106 was studied most extensively. Also, in this case, the CO, hydration activity is drastically reduced but not abolished. Michaelis-Menten behavior was observed. The kmetic parameters obtained under a variety of conditions are given in Table 2. The maximal value of k,,, at alkaline ph is only about 0.1% of that for unmodified enzyme, but the K, values are quite small so that the maximal value of kcatlk,,, is rather large and more than 5% of the value for unmodified enzyme. This behavior strongly suggests that the measured activity is a genuine property of the mutant. In addition, no apparent activation by the buffer 1,Zdimethylimidazole was observed for the mutant in contrast to native human carbonic anhydrase I1 [20]. Instead, the data indicate that this buffer has an inhibitory effect. To avoid buffer inhibition and to facilitate measurements at the low CO, concentration required for good estimates of the small K, most experiments were performed in 5 mm buffer. In crystal structure studies of the mutant with Gln106, extra electron density, probably representing a bound sulfate ion, is observed near the metal ion (Y. Xue, A. Liljas, B.-H. Jonsson and S. Lindskog, unpublished results). This prompted us to test if the concentration of sulfate used in the kinetic experiments for control of ionic strength is sufficient to inhibit the CO, hydration activity. A comparison of data obtained in the absence of sulfate with those obtained in the presence of this anion (Table 2) shows no significant inhibition by sulfate at the highest tested ph (8.8) but a marked inhibition at lower ph. Thus, the inhibition is strongly dependent on ph as illustrated graphically in Fig. 3. Kinetic hydrogen isotope effects were estimated at ph 8.8 (ph meter reading) in 5 mm Taps/NaOH and in the absence of sulfate. The observed effect (value in 'H,O/value in 'H,O) was 1.7 t 0.2 for k,, and for k,,/k,. The CO, hydration activity of the mutant with Ala106 is quite similar to that of the mutant with Gln106 (Table 3). Both these mutants are similarly inhibited by sulfate. Changing Glu106 to Asp has no large effect on the CO, hydration activity. Kinetic parameters obtained at different ph values are given in Table4. For comparison, it can be mentioned that wild-type enzyme in 50 mm Taps/NaOH, ph 8.9, yields ktat = 1000 ms-' and K, = 9.7 mm [20]. Similarly, the substitution of Phe for Tyr7 results in relatively small changes of the kinetic parameters for CO, hydration, as illustrated by the results given in Table 5. Ester hydrolysis The hydrolysis of 4-nitrophenyl acetate catalyzed by the investigated mutants of human carbonic anhydrase I1 was monitored. The activities of the mutants with Ala106, Gln106 and Ala199 were found to be considerably reduced compared to that of unmodified enzyme. The maximal values (at ph 3 8.5) of the apparent second-order rate constant, ken,, are about 30M-'. s-' for the variant with Ala106, 40 M-'. s-' for the variant with Gln106 and 15 M-'. s-' for the variant with Ala199, while the corresponding value for unmodified enzyme is 2800 M-'. s-' [17, 221. On the other hand, the variant with Asp106 gave a maximal ken, of 1800 M-'. s-', whereas the variant with Phe7 is a more active esterase than unmodified enzyme with a maximal ken, of 6300 M-I. s-'. ph dependence of 4-nitrophenyl acetate hydrolase activity of the variants with Phe7, Asp106 and Ala106 is shown in Fig. 4. Inhibitor binding The binding of dansylamide to the mutants with Ala199 and Gln106 was investigated. Binding of this inhibitor to unmodified enzyme leads to an approximately 50-fold increase in the fluorescence intensity of dansylamide and a blue shift of the emission spectrum to 457 nm (Fig. 5). Similar effects are observed when solutions of dansylamide and the mutants are mixed suggesting that the sulfonamide binds to the mutants in a manner similar to the unmodified enzyme. However, the fluorescence intensities of the dansylamide complexes with the mutants are slightly lower than that of the complex with unmodified enzyme. In addition, the blue shifts are significantly smaller. Thus, maximal emission occurs at 466 nm for the mutant with Ala199 and at 474 nm for the mutant with GlnlO6. However, the fluorescence-emission spectra of dansylamide complexes with the mutants with Phe7 and Asp106 were indistinguishable from that of the complex with unmodified enzyme. Spectrofluorimetric titrations were performed to estimate the affinity of dansylamide for the mutants with Ala199 and

4 824 Table 2. Kinetic parameters for CO, hydration catalyzed by human Glu106 + Gln carbonic anhydrase 11. Temperature, 25 "C. DMI, 1,2-dimethylimidazole. A buffer marked by an asterisk indicates that no sulfate was present in the assay medium. In other cases, the ionic strength was kept at 0.1 M by addition of Na,SO,. Parameter values and standard deviations were obtained by non-linear-regression 5 mm Taps* 5 mh4 Taps 50 mm DMI 5 mm DMI 5 mm DMI 5 mm Mops* 10 mm Imidazole 5 mm Mops* 5 mm Mops 5 mm Mes* 5 mm Mes* 5 mm Mes ms-' 1.09? ? ? ? ? ? t t ? t mm 0.11 t t t t ? t t ? t ? 0.5 pm-1. s-1 9.5? ? t t t t t ? t ? Table 3. Kinetic parameters for CO, hydration catalyzed by human Glu106 + Ala carbonic anhydrase 11. Temperature, 25 "C. Buffer concentration, 5 mm. A buffer marked by an asterisk indicates that no sulfate was present in the assay medium. In other cases, the ionic strength was kept at 0.1 M by addition of Na,SO,. Parameter values and standard deviations were obtained by non-linearregression Buffer ph k, Km kc.,lkm ms-' mm pm-'. s-1 Taps t t t 3 Mops* ' ? t 1 Mops ? ? 0.01 Mes* ? t ? 0.03 Mes t I 8 9 PH Fig. 3. ph dependence of k,.,/k,,, for CO, hydration catalyzed by unmodified human carbonic anhydrase I1 and the mutant with Gln106. Temperature, 25 "C; buffer concentration, 5 mm. (A) Unmodified enz$ne in the presence of 33 mm sulfate. The curve was drawn as a simple titration curve with pka = 7.1. (0) Mutant with Gln106 in the absence of sulfate. The curve has been (arbitrarily) drawn as a simple titration curve with pk, = 6.9. (0) Mutant with Gln106 in the presence of 33 mm sulfate. The curve has been drawn as a straight line with a slope of 1. Table 4. Kinetic parameters for CO, hydration catalyzed by human Glu106 + Asp carbonic anhydrase 11. Temperature, 25 "C. Buffer concentration, 50 mm. DMI, 1,2-dimethylirnidazole. The ionic strength was kept at 0.1 M by addition of Na,SO,. Parameter values and standard deviations were obtained by non-linear-regression PH kc,, K, kc,&, ~ ~ f f ~ ~ ms-' mm pm-1. s-1 Taps t t t 4 DMI ? t t 10 Imidazole ? t t 3 Mes t t t 0.4 Gln106. At ph 8.5, the K, for unmodified enzyme is 0.3 pm [23], whereas we obtained K, = 1.2 pm for the Ala199 mutant and 7 pm for the Gln106 mutant. Thus, the binding of dansylamide is only moderately weakened in these mutants. The affinity of acetazolamide (2-acetylamido-l,3,4-thiadiazole-5-sulfonamide) for the mutant with Gln106 was estimated in a competition experiment where the dansylamide/ enzyme complex was titrated with acetazolamide in 20 mm HepesNaOH, ph 8.8. At this ph, K, for dansylamide is also 7 pm, and K, = 2.2 pm was estimated for acetazolamide. Acetazolamide binds to the unmodified enzyme with a K, of about 0.1 pm at similar conditions (S. Lindskog, unpublished data). In the crystal structure of the mutant with Ala199, found an extra electron density near the metal ion is present (Y. Xue, A. Liljas, B.-H. Jonsson and S. Lindskog, unpublished results), and it was proposed to represent a bicarbonate ion. Therefore, the affinities of bicarbonate and another anion, SCN-, were estimated by competition with dansylamide. The results are summarized in Table 6. Human red cell carbonic anhydrase I1 binds bicarbonate with an apparent Ki of about 500 mm [24] and SCN- with K, = 2 mm [25] at ph 7.5.

5 Table 5. Kinetic parameters for CO, hydration catalyzed by human Tyr7 -+ Phe carbonic anhydrase II. Temperature, 25 "C. Buffer concentration, 50 mm. DMI, 1,2-dimethylimidazole. The ionic strength was kept at 0.1 M by addition of Na,SO,. Parameter values and standard deviations were obtained by non-linear-regression Buffer PH k,,, K, kca,jkm ms-l mm pm-1. s-1 Taps ? f 6 DMI f Imidazole f ? f 0.4 Mes f ? I5 f I + - c Ar Wild type Wavelength (nrn) Fig. 5. Fluorescence-emission spectra of dansylamide (DNSA) and its complexes with unmodified human carbonic anhydrase I1 (wild type), the mutant with Ala199 and the mutant with Gln106. Excitation at 320 nm. Dansylamide concentration, 1 pm in all cases. Wild type (10 pm), Glu Gln (E106Q, 40 pm) and free inhibitor in 20 mm Taps/NaOH, ph 8.5. Thr199 + Ala (T199A, 24 pm) in 20 mm Tris/H,SO,, ph 8.5. The ionic strength was adjusted to 0.1 M with NqSO, in all cases. It is estimated that the wild-type enzyme is saturated to 97 % by dansylamide, the mutant with Ala199 to 95% and the mutant with Gln106 to 85%. 'a E106A A I I I PH Fig. 4. pwrate profiles for 4-nitrophenyl acetate hydrolysis catalysed by mutants of human carbonic anhydrase I1 with Phe7 (Y7F), Asp106 (E106D) and Ala106 (E106A). Temperature, 25 "C; the ionic strength was maintained at 0.1 M by addition of Na,SO,. (0) Mutant with Phe7; the curve was constructed as a simple titration curve with pk. = 7.1 and a maximal value of logk,, of (0) Mutant with Aspl06; the curve was constructed as a simple titration curve with pk, = 6.6 and a maximal value of logk.,, of (A) Mutant with Ala106; the curve was calculated assuming a single pk, of 7.9 and logk,,, values of 1.50 and 0.15 for the basic and acidic forms, respectively. In similar experiments with the Gln106 variant at ph 8.0, K, of 11 pm and 1.8 mm were estimated for dansylamide and bicarbonate, respectively, demonstrating enhanced bicarbonate binding also in this variant. DISCUSSION The results of our investigation show clearly that two of the conserved amino acid residues, Thr199 and Glu106, in the hydrogen-bonded network connected to the zinc-bound H,O/OH- are functionally important, whereas Tyr7 is not required for efficient catalysis of CO, hydration or ester hydrolysis. When this study was initiated, all sequenced isoenzyme forms of carbonic anhydrase from a variety of species had tyrosine in sequence position 7. However, it has later turned Table 6. Dissociation constants (KJ for the complexes of dansylamide, bicarbonate and thiocyanate with human Thr199 + Ala carbonic anhydrase 11. Temperature, 20 C. Buffer: Tris/H,SO, with Na,SO, added to yield an ionic strength of 0.1 M. The buffer concentration was 20mM in the case of dansylamide, otherwise 50 mm except for bicarbonate at ph 7.5 when 200 mm buffer was used to avoid significant ph changes. Compound Ki at ph 7.5 ph 8.5 mm Dansylamide SCN HCO, out that Tyr7 is not strictly conserved. Thus, the periplasmic carbonic anhydrase from the unicellular green alga, Chlamydomonas reinhardtii, lacks Tyr7 and has, probably, a deletion in this sequence position [26]. In addition, the amino acid sequence derived from the nucleotide sequence of a cdna from mouse liver, presumably encoding the mitochondrial isoenzyme V, suggests that Tyr7 is replaced by His [27]. In our laboratory, a deletion mutant of human carbonic anhydrase 11, lacking the N-terminal 16 residues (residues 2-17 in the nomenclature used here), has been prepared (G. Aronsson, L.-G. Mhensson and B.-H. Jonsson, unpublished data). Preliminary results indicate that this mutant is almost fully active. Thus, not only Tyr7 but also a sizable segment

6 826 of the polypeptide chain appears to be catalytically redundant. Merz [28] has proposed that an important catalytic function of the Thr199-Glu106 network is to restrict the orientation of zinc-bound OH- so that one of its lone electron pairs is directed towards the CO, molecule located in a hydrophobic pocket in the vicinity [29]. Crystallographic observations of inhibitor [ and HCO, [34] binding to the metal ion have also led to suggestions that Thr199 functions as a door-keeper, restricting access to the metal ion and preferentially selecting hydrogen-bond donors for the tetrahedral coordination site near Thr199 by offering a lone pair of its hydroxyl oxygen as a hydrogen-bond acceptor. In the mutant with Ala199, a metal-bound OH- should not be subjected to the same positional restrictions as in the unmodified enzyme. Data from our crystallographic studies (Y. Yue, A. Liljas, B.-H. Jonsson and L. Lindskog, unpublished results) suggest that the zinc ion in the mutant is still tetracoordinated as in wild-type enzyme, but the metal-bound H,O/OH- has moved from its position in the unmodified enzyme to form a weak hydrogen bond with Glu106 and a van der Waals contact with the methyl group of Ala199. Although the kinetic data (Table l) do not allow firm conclusions as to which step in the catalytic mechanism is most perturbed by the mutation, an upper limit of about 100-fold can be estimated for the effect on the rate constant for the reaction of zinc-bound OH- with CO,. This estimate is based on the assumption that CO, is weakly bound in unmodified enzyme as well as mutant and that CO,/HCO; interconversion rather than HCO; dissociation (cf. Eqn 1) limits the value of k,.jk,,, in the unmodified enzyme. In addition, the pwrate profile for the Ala199 mutant (Fig. 2) suggests that the remarkable acidity of the zincbound water molecule in carbonic anhydrase I1 (pk, = 6.8) to some extent depends on the interaction with Thr199, since removal of this interaction leads to a pk, increase of about 1. This is confirmed by results of J. F. Krebs and C. A. Fierke (personal communication) who found pk, values of 7.3, 8.3, 8.7 and 9.6 for mutants of human carbonic anhydrase I1 with Ser199, Ala199, Val199 and Ro199, respectively. The removal of the y atoms of residue 199 also results in a stronger binding of anions (Table 6), perhaps as a result of relaxed steric restrictions. Part of the difference between the anion affinities of the Ala199 mutant and unmodified enzyme at ph7.5 comes from the different pwrate profiles, but one can estimate that the intrinsic binding affinity for the mutant is enhanced by about 20-fold. Since rapid dissociation of HCO; from the active site is important for efficient catalysis of CO, hydration, one can consider the weakening of the binding of this reaction product as another catalytic role for Thr199. Glu106 acts as a hydrogen-bond acceptor in its interaction with Thr199, thus, restricting the orientation of the hydroxyl group of this residue. When Glu106 is altered to Ala, this restriction as well as a negative charge are removed. Results of crystal-structure studies (Y. Yue, A. Liljas, B.-H. Jonsson and L. Lindskog, unpublished results) suggest that the hydrogen-bonding pattern in the mutant with Gln106 has been reversed so that Gln106 now donates a proton to the hydroxyl group of Thr199. Our data show that this alteration and the loss of a negative charge results in a decrease of sulfonamide affinity by about 20-fold and a very substantial increase in affinity for bicarbonate and sulfate ions. The mutation should, of course, also affect the interaction of the Thr199 hydroxyl group with the metal-bound H,O/OH-. Nevertheless, the maximal values of kjkm measured for the mutants with Ala106 and Gln106 (Tables 2 and 3) are only about one order of magnitude smaller than the kjkm values for unmodified enzyme. The parameter kjkm depends only on rate constants for the steps shown in Eqn (1) [5], whereas k,, also depends on fist-order rate constants from the protontransfer steps required for completion of the catalytic cycle (Eqn 2): E-ZnZ+-H,O + E-Zn2+-OH- + H'. (2) If it is assumed that the observed variations in kjkm reflect changes in COJHCO; interconversion rates at the metal ion center, it seems as if the alteration of residue 106 has no drastic effect on the orientation of the metal-bound OH- by Thr199 or, possibly, the precise orientation of the metal-bound OH- is not as critical as postulated by Merz P81. The substitutions of Ala and Gln for Glu106 have their largest kinetic effects on k,, for CO, hydration. Unfortunately, the data do not allow us to assign these effects conclusively to a particular step in the catalytic mechanisms. The two main candidates are HCO; dissociation and protontransfer steps included in Eqn (2). The observed hydrogenisotope effect of 1.7 is considerably smaller than the effect of 3.8 observed for the rate-limiting proton-transfer step in human red-cell carbonic anhydrase I1 [18], but it seems possible that proton transfer contributes to rate-limitation in these mutants. In this connection, it is interesting to note that Kannan et al. [35] proposed that Glu106 should have a crucial role in this proton-transfer process. Somewhat to our surprise, the substitution of Asp for Glu106 does not result in any large change in catalytic activity. The crystal structure of this mutant reveals that the hydrogen bond between the carboxyl group of residue 106 and Thr199 is preserved. The polypeptide backbone has only moved slightly to compensate for the shorter amino acid side chain, but the side chain of Asp106 has a more extended conformation than the side chain of Glu106 in wild-type enzyme (Y. Yue, A. Liljas, B.-H. Jonsson and L. Lindskog, unpublished results). We are grateful to Dr. Carol A. Fierke for the generous gift of the plasmid paca and to her and Dr. Joseph F. Krebs for communicating unpublished data. We thank Ms. Katarina Wallgren for skillful technical assistance. This work was financially supported by a grant from the Swedish Natural Science Research Council (K 2911). REFERENCES 1. Lindskog, S. & Malmstrom, B. G. (1962) J. Biol. Chem. 237, Coleman, J. E. (1967) Nature 214, Thorslund, A. & Lindskog, S. (1967) Eur. J. Biochem. 3, Eriksson, A. E., Jones, T. A. & Liljas, A. (1988) Proteins Struct. Funct. Genet. 4, Silverman, D. N. & Lindskog, S. (1987) Acc. Chem. Res. 27, Reference deleted. 7. Lindskog, S., Behravan, G., Engstrand, C., Forsman, C., Jonsson, B.-H., Liang, Z., Ren, X. & Xue, Y. (1991) in Carbonic anhydrase. From biochemistry and genetics to physiology and clinical medicine (Botr6, F., Gros, G. & Storey, B. T., eds) pp. 1-13, Verlag Chemie, Weinheim. 8. Lindskog, S. (1983) in Zinc enzymes (Spiro, T. G., ed.) pp , Wiley, New York.

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