We have analyzed the adsorption of protein to the surfaces of silica nanoparticles with diameters of 6, 9, and 15 nm. The effects upon adsorption on variants of human carbonic anhydrase with differing conformational stabilities have been monitored using methods that give complementary information, i.e., circular dichroism (CD), nuclear magnetic resonance (NMR), analytical ultracentrifugation (AUC), and gel permeation chromatography. Human carbonic anhydrase I (HCAI), which is the most stable of the protein variants, establishes a dynamic equilibrium between bound and unbound protein following mixture with silica particles. Gel permeation and AUC experiments indicate that the residence time of HCAI is on the order of approximately 10 min and slowly increases with time, which allows us to study the effects of the interaction with the solid surface on the protein structure in more detail than would be possible for a process with faster kinetics. The effects on the protein conformation from the interaction have been characterized using CD and NMR measurements. This study shows that differences in particle curvature strongly influence the amount of the protein's secondary structure that is perturbed. Particles with a longer diameter allow formation of larger particle-protein interaction surfaces and cause larger perturbations of the protein's secondary structure upon interaction. In contrast, the effects on the tertiary structure seem to be independent of the particles' curvature.

1. The steady-state kinetics of the interconversion of CO, and HCO; catalyzed by human carbonic anhydrase C was studied using 'H,O and ' H 2 0 as solvents. The pH-independent parts of the parameters k,,, and K , are 3 -4 times larger in 'H,O than in ,H,O for both directions of the reaction, while the ratios k,,,/K, show much smaller isotope effects. With either C 0 2 or HCO; as substrate the major pH dependence is observed in k,,,, while K , appears independent of pH. The pK, value characterizing the pH-rate profiles is approximately 0.5 unit larger in 2 H 2 0 than in ' H 2 0 .2. The hydrolysis of p-nitrophenyl acetate catalyzed by human carbonic anhydrase C is approximately 35 faster in 'H20 than in ' H 2 0 . In both solvents the pK, values of the pH-rate profiles are similar to those observed for the C0,-HCO; interconversion.3. It is tentatively proposed that the rate-limiting step at saturating concentrations of C 0 2 or HCO; is an intramolecular proton transfer between two ionizing groups in the active site. It cannot be decided whether the transformation between enzyme-bound CO, and HCO; involves a proton transfer or not.Carbonic anhydrase is a highly efficient catalyst of the reversible interconversion of C 0 2 and HCO,. In a buffered solution not far from neutrality, where Cog-as well as free H + and OH-can be neglected, the stoichiometry of the reaction is CO, + H,O + B e HCO; + BH+, where B and BH' are the basic and acidic buffer components, respectively. Regardless of the specific reaction mechanism, the hydration of C 0 2 must be coupled to the splitting of water, formally into H + and OH-. At some stage of the reaction the OH-ion becomes integrated with C02, while the H + ion ultimately combines with the buffer base. In the reverse reaction OH ~ derived from HCO; must combine with H', originating from the buffer acid, to form H,O. Thus, proton transfers are compulsory ingredients in any mechanism of this reaction.Because of the extremely rapid turnover observed for the enzyme-catalyzed reaction, lo5-lo6 s-' at 25 that H,CO, should be regarded as the substrate species specifically combining with the active site. In effect this means that H + is transported bound to HCO;. It follows from this model that additional proton transfers would have to occur within the enzyme-substrate complex, for example in a concerted reaction as proposed by Kaiser and Lo [3].Arguments against H2C03 as the substrate species combining with the active site have been given by several authors [4-61 pointing out that this would require a second-order rate constant for the binding step exceeding those of diffusion-controlled reactions measured in simpler systems. Alternatively it was suggested that H + is transported between solvent and active site by buffer components acting as proton donors and acceptors. In this case no second-order rate constant involved in the catalytic cycle would have to be greater than lo8-lo9 M-' s -l , and it is not necessary to invoke novel phenomena such as surface diffusion [2,7] to rationalize the e...

The circular dichroism (CD) spectrum of human carbonic anhydrase II (HCAII) has been investigated using various mutants of the enzyme in which tryptophans have been replaced by site-directed mutagenesis. HCAII contains seven tryptophans which are believed to significantly contribute to the CD spectrum in both the near- and far-UV regions. By substituting the tryptophans one at a time, the spectral effects of the individual tryptophans were studied. The near-UV spectrum of HCAII is very complex, with multiple Cotton effects. This complexity has been attributed to aromatic amino acids, especially tryptophans, located in asymmetric aromatic clusters in the molecule. CD spectra of the individual tryptophans were calculated as difference spectra between the CD spectrum of HCAII and those of the tryptophan mutants. These spectra showed that the tryptophans contributed to the CD spectrum in almost the entire wavelength region investigated (180-310 nm). Summation of the individual tryptophan CD spectra in the near-UV region yielded a spectrum that was qualitatively very similar to that of HCAII, showing that the tryptophans are the major determinant for this part of the CD spectrum. Since tryptophans were also demonstrated to contribute significantly in the far-UV region, tryptophans can interfere considerably with the assignment of changes in CD bands to changes in secondary structure content during folding reactions. Moreover, because of this substantial interference, predictions of the amount of various types of secondary structure from CD data from the far-UV region are made more difficult. These findings are probably of general importance for proteins that, like HCAII, contain several tryptophans.(ABSTRACT TRUNCATED AT 250 WORDS)

Several proteins have been discovered that either catalyze slow protein-folding reactions or assist folding in the cell. Prolyl isomerase, which has been shown to accelerate rate-limiting cis-trans peptidyl-proline isomerization steps in the folding pathway, can also participate in the protein-folding process as a chaperone. This function is exerted on an early folding intermediate of carbonic anhydrase, which is thereby prevented from aggregating, whereas the isomerase activity is performed later in the folding process.

The effects of human carbonic anhydrase C on the 13C nuclear magnetic resonance spectra of equilibrium mixtures of l3CO2 and NaH13C03 were measured at 67.89 MHz. Enzyme-catalyzed C02-HCO; exchange rates were estimated from the linewidths of the resonances. The results show that: (a) the maximal exchange rates are larger than the maximal turnover rates; (b) the exchange is equally rapid with 'H2O or with 2H20 as solvents; (c) the exchange is equally rapid in the presence or in the absence of added buffers; (d) the apparent substrate binding is weaker than predicted if steady-state K, values are assumed to represent substrate dissociation constants.The main conclusion concerning the catalytic mechanism of the enzyme is that the protontransfer processes which limit turnover rates in the steady state are not directly involved in C02-HCO: exchange. In addition, the results suggest that COZ-HCO; interconversion takes place by a nucleophilic mechanism, such as a reversible reaction of zinc-coordinated OH-with C02.Carbonic anhydrase is a zinc-containing metalloenzyme catalyzing the reaction C02 + H20 HCO; + H + .( 1) The enzyme is extremely efficient; the turnover number for C 0 2 hydration catalyzed by the human C isoenzyme is lo6 sK1 at 25°C [1,2]. Proton transfer processes are necessarily involved in the reaction. Thus, C02 hydration is associated with the splitting of HzO and the production of H+. The prevalent hypothesis is that rapid H2O splitting is promoted by the metal ion (cf. [3]) which serves as an acceptor for the OH-component of H2O [4]. The H f component of H 2 0 is transferred into the medium perhaps via acceptor groups in the active site [2]. It was pointed out by several authors [l, 5 -81 that a sufficiently rapid release of H' cannot take place unless H' is transported into the medium by some acceptor other than H 2 0 . It was proposed [9-111 that the buffer can have this transport fkction, and this hypothesis has recently gained experimental support [12-141. Thus, at sufficiently low buffer concentration the rate of C02 hydration appears to be limited by a reaction step such as EH + B e E-+ BH'. (2)where B and BH' represent the buffer components [14]. At high buffer concentration reaction 2 is not Abbreviation. NMR, nuclear magnetic resonance. Enzyme. Carbonic anhydrase (EC 4.2.1.1). rate-limiting. However, studies of hydrogen isotope effects on the steady-state kinetic properties of the human C isoenzyme [2] suggested that an intramolecular proton-transfer step separate from the C02-HCO; interconversion limits the rate under these conditions. Schematically, EH' e EH. (3)The presence of such an 'isomerization' step appeared to be supported by product-inhibition patterns [15].To test this hypothetical mechanism further, and to investigate whether proton transfers are involved in the reaction steps associated with substrate-product interconversion, it seemed logical to study the exchange between C02 and HCO; separately. According to our hypothesis this exchange would not involve reactions 2 and 3, but...

Several conformation-sensitive parameters have shown that human carbonic anhydrase II exists as a stable and compact equilibrium folding intermediate of molten globule type. In this study we have continued a previously initiated mapping of the intermediate structure. Cys residues were engineered, one at a time, into various regions of the protein structure, so as to obtain chemically reactive probes and handles for spectroscopic probes. These probes were used to specifically report on conformational changes accompanying the folding process. Thus, the accessibility of the introduced Cys residues to specific chemical labeling by radioactive iodoacetate was used to monitor the stability and compactness of the substructure surrounding each Cys residue. In addition, a spin-label (nitroxide radical) and a fluorescent probe (IAEDANS) were attached to the inserted SH-groups to give complementary information. The mobility of the spin-label was used to indicate local changes in structure, and the fluorophore was used to probe local changes in polarity at various stages of unfolding. Much of the predominant beta-structure, consisting of 10 beta-strands extending throughout the entire molecule, appears to be compact and largely intact in the intermediate. Thus, beta-strands 3-7, probed at positions 68, 97, 118, 123, 206, and 245, seem to have a native-like structure in the folding intermediate. In contrast, a more flexible structure is found around positions 56, 176, and 256 in the peripheral beta-strands 1, 2, and 9, showing that the stability of the secondary structure in the intermediate state is less in the outer parts of the protein. A hydrophobic region, containing beta-strands 3-5, seems to be remarkably stable and is not ruptured until strong denaturing conditions (5 M GuHCl) are applied. The stability of this hydrophobic beta-core appears to increase toward the center. This stable region is contained in the middle of a sequentially continuous antiparallel structure that spans beta-strands 2-6, suggesting that this part might represent a site where folding is initiated.

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.

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