Carbon-13 nuclear magnetic resonance probe of active-site ionizations in human carbonic anhydrase B.

Human carbonic anhydrase B (HCAB), prepared by a new affinity chromatography procedure, was carboxymethylated exclusively at NT of its active-site histidine-200 using 90% [1-13C]bromoacetate. The 13C nuclear magnetic resonance signal of the covalently attached carboxylate was easily detected over the natural abundance background due to the other carbonyl and carboxyl carbons of this 29 000 molecular weight zinc metalloenzyme. Its chemical shift proved very sensitive to the presence of inhibitors in the active site and to variations in pH. Two perturbing groups with pKa values of 6.0 and 9.2 were assigned to the modified histidine-200 itself and the zinc-bound water ligand, respectively, making use of 13C NMR titration data on Nr- and Nr-carboxymethyl-L-histidine model compounds. The results rule out histidine-200 as the critical group whose ionization controls the catalytic activity. They also strongly suggest an interaction of the carboxylate of the carboxymethyl group with either the zinc or its water ligand around pH 8, possibly explaining the basis for the major differences between HCAB and CmHCAB.     

Specificity of interactions of hapten side chains with the combining site of the myeloma protein MOPC 315.

The pKa values of the three histidine residues in the Fv fragment (variable region of the heavy and light chains) of the mouse myeloma protein MOPC 315, measured by high resolution n.m.r. (nuclear magnetic resonance), are 5.9, 6.9 and 8.2. The perturbation of the pKa of one of the histidines (pKa 6.9) on the addition of hapten and the narrow linewidth of its proton resonances suggests that it is at the edge of the combining site. References to the model of the Fv fragment [Padlan, Davies, Pecht, Givol & Wright (1976) Cold Spring Harbor Symp. Quant. Biol. 41, in the press] allows assignment of the three histidine residues, histidine-102H, histidine-97L and histidine-44L. The determination of the pKa of the phosphorus group, by 31P n.m.r., of a homologous series of Dnp- and Tnp- (di- and tri-nitrophenyl) haptens has located a positively charged residue. Molecular-model studies on the conformations of these haptens show that the residue is at the edge of the site. The model suggests that the positively charged residue is either arginine-95L or lysine-52H.     

A totally synthetic histidine-2 ferredoxin: thermal stability and redox properties.

The entire polypeptide of Clostridium pasteurianum ferredoxin (Fd) with a site-substituted tyrosine-2----histidine-2 was synthesized using standard t-Boc procedures, reconstituted to the 2[4Fe-4S] holoprotein, and compared to synthetic C. pasteurianum and native Fds. Although histidine-2 is commonly found in thermostable clostridial Fds, the histidine-2 substitution into synthetic C. pasteurianum Fd did not significantly increase its thermostability. The reduction potential of synthetic histidine-2 Fd was -343 and -394 mV at pH 6.4 and 8.7, respectively, versus standard hydrogen electrode. Similarly, Clostridium thermosaccharolyticum Fd which naturally contains histidine-2 was previously determined to have a pH-dependent reduction potential [Smith, E.T., & Feinberg, B.A. (1990) J. Biol. Chem. 265, 14371-14376]. An electrostatic model was used to calculate the observed change in reduction potential with pH for a homologous ferredoxin with a known X-ray crystal structure containing a hypothetical histidine-2. In contrast, the reduction potential of both native C. pasteurianum Fd and synthetic Fd with the C. pasteurianum sequence was -400 mV versus standard hydrogen electrode and was pH-independent [Smith, E.T., Feinberg, B.A., Richards, J.H., & Tomich, J.M. (1991) J. Am. Chem. Soc. 113, 688-689]. On the basis of the above results, we conclude that the observed pH-dependent reduction potential for both synthetic and native ferredoxins that contain histidine-2 is attributable to the electrostatic interaction between histidine-2 and iron-sulfur cluster II which is approximately 6 A away.     

Correlation proton magnetic resonance studies at 250 MHz of bovine pancreatic ribonuclease. I. Reinvestigation of the histidine peak assignments.

The deuterium exchange kinetics of the C(2) protons of the four histidine residues of native bovine pancreatic ribonuclease A have been followed at pH 6.5 and 8.0 by proton magnetic resonance spectroscopy (1H NMR). Comparison of the order of exchange of the histidine peaks with tritium exchange rates into individual histidine residues [Ohe, M., Matsuo, H., Sakiyama, F., and Narita, K. (1974), J. Biochem. (Tokyo) 75, 1197] supports the previous assignment of histidine NMR peaks H(1) and H(4) to histidine-105 and histidine-48 but requires reassignment of peaks H(2) and H(3) to histidine-119 and histidine-12, respectively. Ribonuclease A samples having differentially deuterated histidines have been used to verify the existence of crossover points in the histidine proton magnetic resonance titration curves and to observe the discontinuous titration curve of histidine-48. Proton magnetic resonance peaks have been assigned to the C(4) protons of the four histidine residues of ribonuclease A on the basis of their unit proton areas and by matching their titration shifts with the more readily visible C(2)-H peaks of the histidines. The pK' values derived from the C(4)-H data agree, within experimental limits, with those derived from C(2)-H data. The C(4)-H peaks were assigned to histidine-12, -48, -105, and -119 of ribonuclease A on the basis of their pH dependence, pK' values, shifts of their pK' values in the presence of inhibitor cytidine 3'-phosphate, and by comparison with the assignments of the histidine C(2)-H peaks above.     

Total synthesis of the structural gene for the precursor of a tyrosine suppressor transfer RNA from Escherichia coli. 11. Enzymatic joining to form the total DNA duplex.

The DNA duplex corresponding to the entire length (126 nucleotides) of the precursor for an Escherichia coli tyrosine tRNA has been synthesized. Duplex [I] (Sekiya, T., Besmer, P., Takeya, T., and Khorana, H. G.(1976) J. Biol. Chem. 251, 634-641), corresponding to the nucleotide sequence 1-26, containing single-stranded ends and carrying one appropriately labeled 5'-phosphate group, was joined to duplex [II] (Loewen, P. C., Miller, R. C., Panet, A., Sekiya, T., and Khorana, H. G. (1976) J. Biol. Chem. 251, 642-650) (nucleotide sequence 23-66 or 23-60) was phosphorylated with [gamma-33P]ATP at the 5'-OH ends. Duplex [III] (Panet, A., Kleppe, R., Kleppe, K., and Khorana, H. G. (1976) J. Biol. Chem. 251, 651-657) (nucleotide sequence 57-94 (Fig. 2)) was also phosphorylated at 5'-ends with [gamma-33P]ATP and was joined to duplex [IV] (Caruthers, M. H., Kleppe, R., Kleppe, K., and Khorana, H. G. (1976) J. Biol. Chem. 251, 658-666) (nucleotide sequence 90-126) which carried a 33P-labeled phosphate group on nucleotide 90. The joined product, duplex [III + IV] (nucleotide sequence 57-126) was characterized. The latter duplex was joined to the duplex [I + II] to give the total duplex. The latter contains singlestranded ends (nucleotides 1 to 6 and 121 to 126) which can either be "filled in" to produce the completely base-paired duplex or may be used to add the promoter and terminator regions at the appropriate ends.     

Interaction of the unique competitive inhibitor imidazole with human carbonic anhydrase B.

Imidazole was previously found to be unique among the inhibitors of human carbonic anhydrase B (HCAB) in that it binds competitively with the CO2 substrate (Khalifah, R. G. (1971), J. Biol. Chem. 246, 2561). We report here an aromatic ultraviolet difference spectral study of its interaction with HCAB and compare it with a variety of other inhibitors. Imidazole is found to be unique in that: (1) it generates a different spectrum upon binding that is also much supressed in intensity; (2) its affinity for HCAB is maximal at high pH, being abolished upon its protonation and being independent of active-site ionizations. Imidazole differs from CO2 in that it binds competitively with the anionic inhibitor iodide. The unique properties of imidazole binding are consistent with the recently determined crystal structure of its complex with HCAB showing it to bind as a weak and distant fifth ligand of the essential zinc atom, rather than displacing the solvent molecule in the fourth ligand position (Kannan, K.K., Petef, M., Fridborg, K., Cid-Dresdner, H., and L?vgren, S. (1977), FEBS Lett 73, 115).     

Exchange of labeled nuclei in the CO2--HCO3--solvent system catalyzed by carbonic anhydrase.

Silverman et al. (1979. J. Am. Chem. Soc. 101:6734-6740) have reported measurements of the loss of 18O to solvent from the isotopically labeled CO2--HCO3-system and of the mixing of 18O and 13C labels within the system, as catalyzed by human carbonic anhydrase C in the pH range 6-8. This work is an extension of earlier work (Silverman and Tu. 1976. J. Am. Chem. Soc. 98:978-984) on the very similar bovine enzyme. The more recent work is analyzed by its authors in terms of the "hydroxide" model for the apparent pH-dependence of enzymatic activity, a model in which the pH-dependence is associated with the presumed ionization of an H2O ligand of the active-site metal ion to OH-. From a comparison of their data with a solution of the coupled differential equations that describe the kinetics of isotope exchange in terms of the model, Silverman et al. derived a pH-dependent rate of exchange for the water molecule which is formed at the active site of the enzyme during dehydration. By contrast, using the same data and a model in which active enzyme has a water molecule on the metal ion at the active site, and similar differential equations, we derive a value for the rate of exchange of water that is pH-independent. This model has the attraction that it explains the magnetic relaxation rate of solvent water protons in the Co2+-substituted enzyme, whereas the hydroxide mechanism cannot explain these data without the introduction of unfounded ad hoc assumptions; further, the presence of an OH- ligand of the metal has never been demonstrated. We also include an analysis of analogous data for the bovine enzyme. One result of our analysis is that the pKa for activity of the enzyme samples used is near 6.0, implying that the bulk of the data were taken when the enzyme was essentially all active. It is straightforward to account for the pH-dependence of the data near and below the pKa by using an empirically-derived value for the pKa. However, we have recently developed a model for the low pH (inactive) enzyme that has been successful in interpreting a wide range of data, and we show that this new view can explain the few points at low pH quite adequately. Additionally, we consider the recent kinetic results for the human C enzyme, obtained at chemical equilibrium by studies of the linewidths of nuclear magnetic resonances of 13C in labeled substrate (Simonsson et al. 1979. Eur. J. Biochem. 93:409-417) and show that these experiments and those of Silverman et al. are all consistent with kinetic data from nonequilibrium stopped-flow experiments, viewed in terms of our model, in the limit of low substrate concentration. Results at higher concentrations indicate that the Michaelis constants and equilibrium constants differ somewhat.     

Magnetic resonance investigation of ionizable residues at the active site of thermolysin.

The details of the pH dependence of the thermodynamic and magnetic interactions of the active-site region of thermolysin in which manganese has replaced the active-site zinc atom and the inhibitor N-trifluoroacetyl-D-phenylalanine have been examined. These show a number of ionizable groups in the active-site region. A cooperative displacement of manganese at the catalytic site is observed as pH is lowered. This appears to be the result of the protonation of histidine-142 and -146 which act as metal ligands. The metal is 50% displaced at pH 6.0. At higher pH values, the environment of the bound manganese changes as a result of the ionization of at least two groups of approximate pKa = 8.5 and 9.5. These values are assigned to tyrosine-157 and to the water molecule which acts as a metal ligand at the active site. The binding behavior of the inhibitor strongly suggests that two molecules of inhibitor bind to the enzyme. The weaker site is competitive with the synthetic substrate FAGLA (furylacryloylglycyl-leucinamide), while the strong site has no effect on FAGLA hydrolysis. This second site is in the vicinity of the active site with a distance of 8 A or less between the trifluoromethyl group and manganese bound at the active site.     

A 13C-n.m.r. investigation of the ionizations within an inhibitor--alpha-chymotrypsin complex. Evidence that both alpha-chymotrypsin and trypsin stabilize a hemiketal oxyanion by similar mechanisms.

13C-n.m.r. was used to investigate the structure of the inhibitor enzyme complex formed when alpha-chymotrypsin is alkylated by L-1-chloro-4-phenyl-3-tosylamido-[2-13C]butan-2-one. Two signals are detected. The one at 204.82 +/- 0.11 p.p.m. does not titrate from pH 3 to 9 and is assigned to alkylated methionine-192. The second signal titrates from 99.08 p.p.m. to 103.44 p.p.m. with pKa 8.67. This signal is assigned to a tetrahedral adduct formed between the hydroxy group of serine-195 and the inhibitor. The titration shift of the tetrahedral adduct is ascribed to the ionization of the hemiketal hydroxy group. It is proposed that the resulting oxyanion is stabilized by interaction with the imidazolium ion of histidine-57. It is argued that this interaction must raise the pKa of at least 70% of histidine-57 to greater than 11. On denaturation/autolysis of the inhibitor-enzyme complex neither of the signals associated with the intact complex is detected, but a new signal is observed that titrates from 203.52 p.p.m. to 206.08 p.p.m. with pKa = 5.27. This titration shift is assigned to the ionization of the imidazolium ion of alkylated histidine, confirming that the inhibitor has alkylated histidine-57. The significance of these results for the catalytic mechanism of the serine proteinases is discussed.     

The interaction of acetate and formate with cobalt carbonic anhydrase. An NMR study.

The interaction of formate and acetate ions with cobalt-substituted carbonic anhydrase (CA) has been investigated through 13C-NMR and one-dimensional and two-dimensional 1H-NMR spectroscopy. 13C data on formate are consistent with a regularly coordinated ligand, as previously proposed for the acetate anion [Bertini, I., Luchinat, C. & Scozzafava, A. (1977) J. Chem. Soc. Dalton Trans., 1962-1965]. 1H-NOE experiments on both anions give evidence of through-space interactions between ligand protons and protein protons. The latter are assigned to specific residues in the active cavity through nuclear Overhauser effect spectroscopy (NOESY) experiments. The 13C-derived and 1H-derived constrains allow reliable docking of these ligands in the active-site cavity. The resulting geometries are similar to one another and consistent with five-coordinated structures around the metal ion, as previously proposed from electronic spectroscopy [Bertini, I., Canti, G., Luchinat, C. & Scozzafava, A. (1978) J. Am. Chem. Soc. 100, 4873-4877]. The results are discussed in light of the current debate on anion binding to metal ions in carbonic anhydrase [Lindahl, M., Svensson, A. & Liljas, A. (1992) Proteins, in the press]; Bertini, I., Luchinat, C., Pierattelli, R. & Vila, A. J. (1992) Inorg. Chem., in the press; Banci, L. & Merz, K. (1992) unpublished results] and, in particular, of the proposed long Zn-O distance found in the recent X-ray results on the formate adduct [Hakanson, K., Carlsson, M., Svensson, A. & Liljas, A. (1992) J. Mol. Biol., in the press].     

Characterization of the histidine proton nuclear magnetic resonances of a semisynthetic ribonuclease

The proton magnetic resonance spectrum at 300 MHz of the histidine residues in a semisynthetic derivative of bovine pancreatic ribonuclease (RNase A) has been determined. The derivative RNase 1-118 . 111-124 was prepared by enzymically removing six residues from the COOH terminus of the protein (positions 119-124) and then complementing the inactive RNase 1-118 with a chemically synthesized peptide containing the COOH-terminal 14 residues of ribonuclease (RNase 111-124) [Lin, M.C., Gutte, B., Moore, S., & Merrifield, R.B. (1970) J. Biol. Chem. 245, 5169-5170]. Comparison of the line positions of the C(2)-1H resonances of these residues and of their pH dependence with those reported by other workers has allowed assignment of the resonances to individual residues, as well as the determination of individual pK values for histidine-12, histidine-105, and histidine-119. The assignment of histidine-119 was confirmed by the use of a selectively deuterated derivative. The titration behavior of all four histidine residues is indistinguishable from that observed by others for bovine pancreatic ribonuclease A. Partial dissociation of the noncovalent semisynthetic complex was evident at 30 degrees C, pH 4.0, 0.3 M NaCl; pertinent spectra were analyzed to provide an estimate of the association constant between the component chains under these conditions of 1.9 X 10(3) M-1.     

Interaction of phosphate analogues with glyceraldehyde-3-phosphate dehydrogenase.

The glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase catalyzes the oxidative phosphorylation of D-glyceraldehyde 3-phosphate. A variety of phosphonates have been shown to substitute for phosphate in this reaction [Gardner, J. H., & Byers, L. D., (1977) J. Biol. Chem. 252, 5925--5927]. The dependence of the logarithm of the equilibrium constant for the reaction on the pKa2 value of the phosphonate is characterized by a Br?nsted coefficient, betaeq, of approximately 1. This represents the sensitivity of the transfer of the phosphoglyceroyl group between the active-site sulfhydryl residue (in the acyl-enzyme intermediate) and the acyl acceptor on the basicity of the acyl acceptor. Molybdate (MoO42-) can also serve as an acyl acceptor in the glyceraldehyde-3-phosphate dehydrogenase catalyzed reaction. The second-order rate constant for the reaction with molybdate is only approximately 12 times lower than the reaction with phosphate even though the pKa2 of molybdate is 3.1 units lower than the pKa2 of phosphate. The immediate product of the molybdate reaction is the acyl molybdate, 1-molybdo-3-phosphoglycerate. The acyl molybdate, like the acyl arsenate (the immediate product of the reaction when arsenate is the acyl acceptor), is kinetically unstable. At pH 7.3 (25 degrees C), the half-life for hydrolysis of the acyl molybdate, or the acyl arsenate, is less than 2.5 s. Thus, hydrolysis of 1-molybdo- and 1-arseno-3-phosphoglycerate is at least 2000 times faster than hydrolysis of 1,3-diphosphoglycerate under the same conditions. Glyceraldehyde-3-phosphate dehydrogenase has a fairly broad specificity for acyl acceptors. Most tetrahedral oxy anions tested are substrates for the enzyme (except SO4(2-) and SeO4(2-)). Tetrahedral monoanions such as ReO4- and GeO(OH)3- are not substrates but do bind to the enzyme. These results suggest the requirement of at least one anionic site on the acyl acceptor required for binding and another anionic group on the acyl receptor required for nucleophilic attack on the acyl enzyme.     

Alternate substrates of dopamine beta-hydroxylase. I. Kinetic investigations of benzyl cyanides as substrates and inhibitors

A series of benzyl cyanide analogs have been studied as substrates and inhibitors of dopamine beta-hydroxylase to extend our initial report (Baldoni, J. M., and Villafranca, J. J. (1980) J. Biol. Chem. 255, 8987-8990) which showed that p-hydroxybenzyl cyanide was a suicide substrate of dopamine beta-hydroxylase. Thus, the appVmax values for benzyl cyanide analogs decrease in the order p-OH greater than m-OH greater than H much greater than p-OCH3,m-OCH3; the m-OH, m-OCH3 and p-OCH3 analogs are competitive inhibitors versus tyramine in initial velocity studies. The Vmax values for tyramine and p-hydroxybenzyl cyanide are nearly identical at saturating O2 and ascorbate (pH 5.0, 37 degrees C) but the Km for O2 is 0.14 and 2.8 mM, respectively, with tyramine and p-hydroxybenzyl cyanide. Studies of the pH dependence of log V/K for tyramine show two pKa values of 5.2 and 5.8 while for m-hydroxybenzyl cyanide the values are 5.3 and 5.9. The log Vmax profile shows one pKa of 5.9 with tyramine as substrate. Thus, nearly identical enzymic groups are involved in binding and/or catalysis with these two substrates. All the benzyl cyanide analogs are suicide inactivators of dopamine beta-hydroxylase. With m-hydroxybenzyl cyanide, the partition between catalysis and inactivation (kcat/kinact) changed from approximately 600 to approximately 17 as the pH varied from 5.0 to 6.7. The log kinact versus pH profile shows one pKa value of 6.0, suggesting that an enzymic group must be deprotonated for maximal inactivation. Copper was essential for the suicide inactivation of dopamine beta-hydroxylase by benzyl cyanides and kinetic studies of partially inhibited dopamine beta-hydroxylase (approximately 50%) showed that inactive enzyme molecules were completely inactive. The following papers in this series discuss the partial reactivation of suicide-inhibited dopamine beta-hydroxylase and the stoichiometry of inactivation by benzyl cyanide analogs.     

A 13C-n.m.r. investigation of ionizations within a trypsin-inhibitor complex. Evidence that the pKa of histidine-57 is raised by interaction with the hemiketal oxyanion.

The 13C-n.m.r. titration shifts of the alpha-methylene group of N-alkylated imidazoles are shown to be a sensitive probe of the ionization of the imidazolium ion. The 13C-n.m.r. titration shifts of both the intact and denatured/autolysed 2-13C- and 1-13C-enriched trypsin-7-amino-3-benzyloxycarbonylamino-1-chloroheptan-2-one (Z-Lys-CH2Cl) complexes are compared. The titration shift for the denatured/autolysed complex confirms that this ionization is due to deprotonation of the N-alkylated imidazolium ring of histidine-57. In the intact trypsin-inhibitor complex the titration shift due to the 1-13C-enriched carbon is anomalous. We conclude that this titration shift cannot arise solely from the ionization of the imidazolium ion of histidine-57 and that the pKa of the imidazolium ion of histidine-57 is raised in the trypsin-inhibitor complex. The relevance of these studies to the mechanism of action of the serine proteinases is discussed.     

Dihydrofolate reductase. The stereochemistry of inhibitor selectivity

X-ray structural results are reported for 10 triazine and pyrimidine inhibitors of dihydrofolate reductase, each one studied as a ternary complex with NADPH and chicken dihydrofolate reductase. Analysis of these data and comparison with structural results from the preceding paper (Matthews, D.A., Bolin, J.T., Burridge, J.M., Filman, D.J., Volz, K.W., Kaufman, B. T., Beddell, C.R., Champness, J.N., Stammers, D.K., and Kraut, J. (1985) J. Biol. Chem. 260, 381-391) in which we contrasted binding of the antibiotic trimethoprim (TMP) to chicken dihydrofolate reductase on the one hand with its binding to Escherichia coli dihydrofolate reductase on the other, permit identification of differences that are important in accounting for TMP's selectivity. The crystallographic evidence strongly suggests that loss of a potential hydrogen bond between the 4-amino group of TMP and the backbone carbonyl of Val-115 when TMP binds to chicken dihydrofolate reductase but not when it binds to the E. coli reductase is the major factor responsible for this drug's more potent inhibition of bacterial dihydrofolate reductase. A key finding of the current study which is important in understanding why TMP binds differently to chicken and E. coli dihydrofolate reductases is that residues on opposite sides of the active-site cleft in chicken dihydrofolate reductase are about 1.5-2.0 A further apart than are structurally equivalent residues in the E. coli enzyme.     

13C NMR study of the ionizations within a trypsin-chloromethyl ketone inhibitor complex

13C NMR is used to detect ionizations within a trypsin-chloromethyl ketone inhibitor complex. The pKa values observed are compared with those predicted by free-energy relationships. For the denatured/autolyzed inhibitor complex, a pKa = 5.26 is observed, which is assigned to the ionization of the imidazole of histidine-57. For the intact inhibitor complex a pKa = 7.88 is determined. This pKa is assigned to the ionization of the hemiketal hydroxyl (pKa = 7.88-8.1) and provides the first direct evidence that the serine proteases are able to stabilize the oxyanion of tetrahedral adducts. Indirect evidence is adduced that the imidazole pK1 of histidine-57 is greater than or equal to 8.1. Line-broadening studies suggest that there may be extra fast exchange line broadening, which could result from rapid tautomeric exchange between neutral and zwitterionic species within the inhibitor complex. The significance of these results for the catalytic mechanism of serine proteases is discussed.     

Catalytic mechanism of scytalone dehydratase: site-directed mutagenisis, kinetic isotope effects, and alternate substrates.

On the basis of the X-ray crystal structure of scytalone dehydratase complexed with an active center inhibitor [Lundqvist, T., Rice, J., Hodge, C. N., Basarab, G. S., Pierce, J. and Lindqvist, Y. (1994) Structure (London) 2, 937-944], eight active-site residues were mutated to examine their roles in the catalytic mechanism. All but one residue (Lys73, a potential base in an anti elimination mechanism) were found to be important to catalysis or substrate binding. Steady-state kinetic parameters for the mutants support the native roles for the residues (Asn131, Asp31, His85, His110, Ser129, Tyr30, and Tyr50) within a syn elimination mechanism. Relative substrate specificities for the two physiological substrates, scytalone and veremelone, versus a Ser129 mutant help assign the orientation of the substrates within the active site. His85Asn was the most damaging mutation to catalysis consistent with its native roles as a general base and a general acid in a syn elimination. The additive effect of Tyr30Phe and Tyr50Phe mutations in the double mutant is consistent with their roles in protonating the substrate's carbonyl through a water molecule. Studies on a synthetic substrate, which has an anomeric carbon atom which can better stabilize a carbocation than the physiological substrate (vermelone), suggest that His110Asn prefers this substrate over vermelone in order to balance the mutation-imposed weakness in promoting the elimination of hydroxide from substrates. All mutant enzymes bound a potent active-site inhibitor in near 1:1 stoichiometry, thereby supporting their active-site integrity. An X-ray crystal structure of the Tyr50Phe mutant indicated that both active-site waters were retained, likely accounting for its residual catalytic activity. Steady-state kinetic parameters with deuterated scytalone gave kinetic isotope effects of 2.7 on kcat and 4.2 on kcat/Km, suggesting that steps after dehydration partially limit kcat. Pre-steady-state measurements of a single-enzyme turnover with scytalone gave a rate that was 6-fold larger than kcat. kcat/Km with scytalone has a pKa of 7.9 similar to the pKa value for the ionization of the substrate's C6 phenolic hydroxyl, whereas kcat was unaffected by pH, indicating that the anionic form of scytalone does not bind well to enzyme. With an alternate substrate having a pKa above 11, kcat/Km had a pKa of 9.3 likely due to the ionization of Tyr50. The non-enzyme-catalyzed rate of dehydration of scytalone was nearly a billion-fold slower than the enzyme-catalyzed rate at pH 7.0 and 25 degrees C. The non-enzyme-catalyzed rate of dehydration of scytalone had a deuterium kinetic isotope effect of 1.2 at pH 7.0 and 25 degrees C, and scytalone incorporated deuterium from D2O in the C2 position about 70-fold more rapidly than the dehydration rate. Thus, scytalone dehydrates through an E1cb mechanism off the enzyme.     

Direct measurement of the pKa of aspartic acid 26 in Lactobacillus casei dihydrofolate reductase: implications for the catalytic mechanism.

The ionization state of aspartate 26 in Lactobacillus casei dihydrofolate reductase has been investigated by selectively labeling the enzyme with [13Cgamma] aspartic acid and measuring the 13C chemical shifts in the apo, folate-enzyme, and dihydrofolate-enzyme complexes. Our results indicate that no aspartate residue has a pKa greater than approximately 4.8 in any of the three complexes studied. The resonance of aspartate 26 in the dihydrofolate-enzyme complex has been assigned by site-directed mutagenesis; aspartate 26 is found to have a pKa value of less than 4 in this complex. Such a low pKa value makes it most unlikely that the ionization of this residue is responsible for the observed pH profile of hydride ion transfer [apparent pKa = 6.0; Andrews, J., Fierke, C. A., Birdsall, B., Ostler, G., Feeney, J., Roberts, G. C. K., and Benkovic, S. J. (1989) Biochemistry 28, 5743-5750]. Furthermore, the downfield chemical shift of the Asp 26 (13)Cgamma resonance in the dihydrofolate-enzyme complex provides experimental evidence that the pteridine ring of dihydrofolate is polarized when bound to the enzyme. We propose that this polarization of dihydrofolate acts as the driving force for protonation of the electron-rich O4 atom which occurs in the presence of NADPH. After this protonation of the substrate, a network of hydrogen bonds between O4, N5 and a bound water molecule facilitates transfer of the proton to N5 and transfer of a hydride ion from NADPH to the C6 atom to complete the reduction process.     

Zymogen activation in serine proteinases. Proton magnetic resonance pH titration studies of the two histidines of bovine chymotrypsinogen A and chymotrypsin Aalpha.

Reversible unfolding of bovine chymotrypsinogen A in 2H2O either by heating at low pH or by exposure to 6 M guanidinium chloride results in the exchange of virtually all the nitrogen-bound hydrogens that give rise to low-field 1H NMR peaks, without significant exchange of the histidyl ring Cepsilon1 hydrogens. These preexchange procedures have enabled the resolution of two peaks, using 250-MHz correlation 1H NMR spectroscopy, that are attributed to the two histidyl residues of chymotrypsinogen A. Assignments of the Cepsilon1 hydrogen peaks to histidine-40 and -57 were based on comparison of the NMR titration curves of the native zymogen with those of the diisopropylphosphoryl derivative. Two histidyl Cepsilon1 H peaks were also resolved with solutions of preexchanged chymotrypsin Aalpha. The histidyl peaks of chymotrypsin Aalpha were assigned by comparison of NMR titration curves of the free enzyme with those of its complex with bovine pancreatic trypsin inhibitor (Kunitz). The NMR titration curves of histidine-57 in the zymogen and enzyme and histidine-40 in the zymogen exhibit two inflections; the additional inflections were assigned to interactions with neighboring carboxyl groups: aspartate-102 in the case of histidine-57 and aspartate-194 in the case of histidine-40 of the zymogen. In bovine chymotrypsinogen A in 2H2O at 31 degrees C, histidine-57 has a pK' of 7.3 and aspartate-102 a pK' of 1.4, and the histidine-40-aspartate-194 system exhibits inflections at pH 4.6 and 2.3. In bovine chymotrypsin Aalpha under the same conditions, the histidine-57-aspartate-102 system has pK' values of 6.1 and 2.8, and histidine-40 has a pK' of 7.2. The results suggest that the pK' of histidine-57 is higher than the pK' of aspartate-102 in both zymogen and enzyme. A significant difference exists in the structure and properties of the catalytic center between the zymogen and activated enzyme. In addition to the difference in pK' values, the chemical shift of histidine-57, which is highly abnormal in the zymogen (deshielded by 0.6 ppm), becomes normalized upon activation. These changes may explain part of the increase in the catalytic activity upon activation. The 1H NMR chemical shift of the Cepsilon1 H of histidine-57 in the chymotrypsin Aalpha-pancreatic trypsin inhibitor (Kunitz) complex is constant between pH 3 and 9 at a value similar to that of histidine-57 in the porcine trypsin-pancreatic trypsin inhibitor complex [Markley, J.L., and Porubcan, M. A. (1976), J. Mol. Biol. 102, 487--509], suggesting that the mechanisms of interaction are similar in the two complexes.