NMR study of the active site of resting state and cyanide-inhibited lignin peroxidase from Phanerochaete chrysosporium. Comparison with horseradish peroxidase

One- and two-dimensional 1H NMR spectroscopy has been used to probe the active site of the high spin ferric resting state and the low spin, cyanide-inhibited derivative of isozyme H2 of the lignin peroxidase, LiP, from Phanerochaete chrysosporium strain BKM 1767. One-dimensional NMR revealed a resting state LiP that is five coordinate at 25 degrees C with an electronic structure similar to that of horseradish peroxidase, HRP. Differential paramagnetic relaxivity was used to identify the C beta H signals of the axial His177. A combination of bond correlation spectroscopy and nuclear Overhauser effect spectroscopy of cyanide-inhibited LiP (LiP-CN) has allowed the assignment of all resolved heme resonances without recourse to isotope labeling, as well as those of the proximal His177 and the distal His48. The surprising effectiveness of the two dimensional NMR methods on such a large and paramagnetic protein indicates that such two dimensional experiments can be expected to have major impact on solution structure determination of diverse classes of heme peroxidases. The two dimensional NMR data of LiP-CN reveal a heme contact shift pattern that reflects a close similarity to that of HRP-CN, including the unusual in-plane trans and cis orientation of the 2- and 4-vinyls. The axial His177 also exhibits the same orientation relative to the heme as in HRP-CN. The proximal His177 contact shifted resonances of both the low spin LiP-CN and high spin LiP are shown to reflect significantly reduced hydrogen bond donation by, or imidazolate character for, the axial histidine in LiP relative to HRP, which may explain the higher redox potential of LiP. The signals are identified for a distal residue that originates from the protonated His48 with disposition relative to the heme similar to that found for the distal His42 in HRP-CN. In contrast, the absence of any resolved signals attributable to an Arg44 in LiP-CN suggest that this distal residue has an altered orientation relative to the heme compared with that of the conserved Arg38 in HRP-CN (Thanabal, V., de Ropp, J. S., and La Mar, G. N. (1987) J. Am. Chem. Soc. 109, 7516-7525).     

Kinetic isotope effect and the presteady-state kinetics of the reaction catalyzed by the bacterial formate dehydrogenase

The primary kinetic isotope effect of the reaction catalyzed by NAD+-dependent formate dehydrogenase (EC 1.2.1.2.) from the methylotrophic bacterium Pseudomonas sp. 101 has been studied. Analysis of the ratios HVm/DVm and H(Vm/KM)/D(Vm/KM) in the pH range 6.1-7.9 showed that the transfer of hydride ion in ternary enzyme-substrate complex is a limiting step of the reaction, and the formate binding to the binary complex (formate dehydrogenase + NAD+) reached equilibrium when the pH of the medium was increased. An approach has been developed to determine the elementary constants of substrate association (kon) and dissociation (koff) at the stages of the binary--ternary enzyme-substrate complexes for the random equilibrium 2-substrate kinetic mechanism. The kon and koff values obtained for the bacterial formate dehydrogenase by using the proposed approach for NAD+ were (4.8 +/- 0.8)*10(5)M-1s-1 and (90 +/- 10) s-1, and for formate (2.0 +/- 1.0)*10(4) M-1s-1 and (60 +/- 20) s-1, respectively.     

Kinetics of the formation and isomerization of methotrexate complexes of recombinant human dihydrofolate reductase

The kinetics of inhibitor binding to highly purified recombinant human dihydrofolate reductase (rHDHFR) have been examined. Methotrexate (MTX) binds rapidly (kon = 1.0 x 10(8) M-1 s-1) and tightly (koff/kon = 210 pM) to the preformed complex of rHDHFR with NADPH. The initial association reaction between rHDHFR.NADPH and MTX is followed by an isomerization of the resulting complex (kiso = 0.4 s-1) leading to a new conformer in which MTX is bound even more tightly (Ki = 3.4 pM). Similar results have been obtained with a major metabolite of MTX having four additional glutamate residues for which Ki = 1.4 pM. 7-HydroxyMTX, another major metabolite of MTX, is a weak inhibitor of rHDHFR (Ki = 8.9 nM), and a polyglutamate form of this metabolite is an equally weak inhibitor (Ki = 9.9 nM), so that the addition of glutamate residues to MTX or 7-hydroxyMTX has little effect on their binding. It follows that the significance of MTX polyglutamate formation relates to other roles such as increasing the cytotoxicity of MTX by prolonging intracellular retention of the drug. Another antifolate, trimethoprim, binds tightly to dihydrofolate reductases from bacterial sources, but weakly to rHDHFR in the ternary complex (KD = 0.5 microM). Although the association step is rapid (kon = 0.4 x 10(8) M-1 s-1), the dissociation rate is also rapid (koff = 15 s-1). Furthermore, there is no isomerization of the ternary complex of trimethoprim with rHDHFR, in contrast to the known isomerization of complexes of trimethoprim with bacterial dihydrofolate reductases.     

Mutation of residues critical for benzohydroxamic acid binding to horseradish peroxidase isoenzyme C.

Aromatic substrate binding to peroxidases is mediated through hydrophobic and hydrogen bonding interactions between residues on the distal side of the heme and the substrate molecule. The effects of perturbing these interactions are investigated by an electronic absorption and resonance Raman study of benzohydroxamic acid (BHA) binding to a series of mutants of horseradish peroxidase isoenzyme C (HRPC). In particular, the Phe179 --> Ala, His42 --> Glu variants and the double mutant His42 --> Glu:Arg38 --> Leu are studied in their ferric state at pH 7 with and without BHA. A comparison of the data with those previously reported for wild-type HRPC and other distal site mutants reaffirms that in the resting state mutation of His42 leads to an increase of 6-coordinate aquo heme forms at the expense of the 5-coordinate heme state, which is the dominant species in wild-type HRPC. The His42Glu:Arg38Leu double mutant displays an enhanced proportion of the pentacoordinate heme state, similar to the single Arg38Leu mutant. The heme spin states are insensitive to mutation of the Phe179 residue. The BHA complexes of all mutants are found to have a greater amount of unbound form compared to the wild-type HRPC complex. It is apparent from the spectral changes induced on complexation with BHA that, although Phe179 provides an important hydrophobic interaction with BHA, the hydrogen bonds formed between His42 and, in particular, Arg38 and BHA assume a more critical role in the binding of BHA to the resting state.     

1H NMR study of the effect of variable ligand on heme oxygenase electronic and molecular structure

Heme oxygenase carries out stereospecific catabolism of protohemin to yield iron, CO and biliverdin. Instability of the physiological oxy complex has necessitated the use of model ligands, of which cyanide and azide are amenable to solution NMR characterization. Since cyanide and azide are contrasting models for bound oxygen, it is of interest to characterize differences in their molecular and/or electronic structures. We report on detailed 2D NMR comparison of the azide and cyanide substrate complexes of heme oxygenase from Neisseria meningitidis, which reveals significant and widespread differences in chemical shifts between the two complexes. To differentiate molecular from electronic structural changes between the two complexes, the anisotropy and orientation of the paramagnetic susceptibility tensor were determined for the azide complex for comparison with those for the cyanide complex. Comparison of the predicted and observed dipolar shifts reveals that shift differences are strongly dominated by differences in electronic structure and do not provide any evidence for detectable differences in molecular structure or hydrogen bonding except in the immediate vicinity of the distal ligand. The readily cleaved C-terminus interacts with the active site and saturation-transfer allows difficult heme assignments in the high-spin aquo complex.     

Inhibition of pig liver esterase by trifluoromethyl ketones: modulators of the catalytic reaction alter inhibition kinetics

The kinetics of substrate hydrolysis by pig liver esterase show activation by various substrates as well as activation by organic solvents (both Vmax and Km increase) [Barker, D.L., & Jencks, W.P. (1969) Biochemistry 8, 3890]. The trifluoromethyl ketones 1,1,1-trifluoro-4-phenylbutan-2-one (TPB) and 1,1,1-trifluoro-4-(p-hydroxyphenyl)butan-2-one (OH-TPB) are slow, tight binding inhibitors of pig liver esterase with Ki values of 6.8 X 10(-9) M and 6.0 X 10(-9) M, respectively. Acetonitrile, TPB, and OH-TPB as well as the substrates pNPA and ethyl lactate caused a 15-130-fold increase in the rate of association (kon), and dissociation (koff), of the enzyme--TPB complex. The value of Ki (koff/kon) did not change. The effect cannot be attributed to half-sites reactivity since an increase in koff of OH-TPB is also observed with enzyme monomers. The results are consistent with a model proposed for the catalytic reaction (Barker & Jencks, 1969) which invokes two binding sites on each esterase subunit, a catalytic site and an effector site. Occupation of the effector site can increase koff and kon for the inhibitors TPB and OH-TPB. Not all compounds which bind at the effector site increase koff. Butanol binds at the effector site but does not effect koff of TPB. The results also indicate that an aromatic or a hydrophobic structure and a carbonyl group are required for optimal interaction with the effector site.     

Mechanism of enantioselective oxygenation of sulfides catalyzed by chloroperoxidase and horseradish peroxidase. Spectral studies and characterization of enzyme-substrate complexes.

The binding of a series of alkyl aryl sulfides to chloroperoxidase (CPO) and horseradish peroxidase (HRP) has been investigated by optical difference spectroscopy, circular dichroism, paramagnetic NMR spectroscopy, and NMR relaxation measurements. The data are consistent with binding of the sulfides in the distal side of the heme pocket with CPO and near the heme edge with HRP. A linear correlation between the binding constants of para-substituted sulfides to CPO and the Taft sigma I parameter suggests that these substrates act as donors in donor-acceptor complexes involving some residue of the protein chain. Spectral studies during turnover show that high enantioselectivity in the CPO-catalyzed oxidation of sulfides results from a reaction pathway that does not involve the accumulation of compound II enzyme intermediate.     

Role of aspartate 27 in the binding of methotrexate to dihydrofolate reductase from Escherichia coli

Dihydrofolate reductase from wild-type Escherichia coli (WT-ECDHFR) and from a mutant enzyme in which aspartate 27 is replaced by asparagine have been compared with respect to the binding of the inhibitor methotrexate (MTX). Although the Asp27----Asn substitution causes only small changes in the association rate constants (kon) for the formation of binary and ternary (with NADPH) complexes, the dissociation rate constants for these complexes (koff) are increased for the mutant enzyme by factors of about 5- and 100-fold, respectively, at pH 7.65. In binding experiments, the initial MTX binary and ternary complexes of the mutant enzyme were found to undergo relatively rapid isomerization (kobs approximately 17 and 145 s-1, respectively). Although such rapid isomerization of complexes of WT-ECDHFR could not be detected in binding experiments, evidence of a slow isomerization (k = 4 x 10(-3) s-1) of the ternary WT-ECDHFR.MTX.NADPH complex was obtained from progress of inhibition experiments. This slow isomerization increases binding of MTX to WT-ECDHFR only 2.4-fold (much less than previously estimated). From presently available data, we could not determine the contribution of the rapid isomerization of complexes to the binding of MTX to the mutant enzyme. The Asp27----Asn substitution increases the overall dissociation constant (KD) 9-fold for the binary complex and 85-fold for the ternary complex. When it is also taken into account that a proton ultimately derived from the solvent must be added to MTX bound to the WT enzyme, but not to MTX bound to the mutant enzyme, these increases in KD for the mutant enzyme correspond to decreases in binding energy for MTX of 3.9 and 5.2 kcal/mol at pH 7.65 for the binary and ternary complexes, respectively.     

Thermodynamic analysis of the binding of aromatic hydroxamic acid analogues to ferric horseradish peroxidase.

Peroxidases typically bind their reducing substrates weakly, with K(d) values in the millimolar range. The binding of benzhydroxamic acid (BHA) to ferric horseradish peroxidase isoenzyme C (HRPC) [K(d) = 2.4 microM; Schonbaum, G. R. (1973) J. Biol. Chem. 248, 502-511] is a notable exception and has provided a useful tool for probing the environment of the peroxidase aromatic-donor-binding site and the distal heme cavity. Knowledge of the underlying thermodynamic driving forces is key to understanding the roles of the various H-bonding and hydrophobic interactions in substrate binding. The isothermal titration calorimetry results of this study on the binding of aromatic hydroxamic acid analogues to ferric HRPC under nonturnover conditions (no H(2)O(2) present) confirm the significance of H-bonding interactions in the distal heme cavity in complex stabilization. For example, the binding of BHA to HRPC is enthalpically driven at pH 7.0, with the H-bond to the distal Arg38 providing the largest contribution (6.74 kcal/mol) to the binding energy. The overall relatively weak binding of the hydroxamic acid analogues to HRPC is due to large entropic barriers (-11.3 to -37.9 eu) around neutral pH, with the distal Arg38 acting as an "entropic gate keeper". Dramatic enthalpy-entropy compensation is observed for BHA and 2-naphthohydroxamic acid binding to HRPC at pH 4.0. The enthalpic loss and entropic gain are likely due to increased flexibility of Arg38 in the complexes at low pH and greater access by water to the active site. Since the Soret absorption band of HRPC is a sensitive probe of the binding of hydroxamic acids and their analogues, it was used to investigate the binding of six donor substrates over the pH range of 4-12. The negligible pH dependence of the K(d) values corrected for substrate ionization suggests that enthalpy-entropy compensation is operative over a wide pH range. Examination of the thermodynamics of binding of ring-substituted hyrazides to HRPC reveals that the binding affinities of aromatic donors are highly sensitive to the position and nature of the ring substituent.     

Resonance Raman investigation of ferric iron in horseradish peroxidase and its aromatic donor complexes at room and low temperatures

Resonance Raman (RR) spectra of the acidic form of FeIII horseradish peroxidase (HRP) were obtained at room and low temperatures using B- and Q-band excitation. At 296 K, HRP exhibits two sets of porphyrin skeletal stretching frequencies which are attributed to a thermal mixture of 5- and 6-coordinate high-spin FeIII states. When the temperature is lowered, the observed bands shift to higher frequencies, and these are assigned to intermediate- and low-spin states. Addition of 40% glycerol has no effect on the spectra at 296 K, but at 20 K, all four frequency sets are observed corresponding to the two forms observed at room and low temperature in the absence of glycerol. The 296 K RR spectrum of the HRP-hydroquinone complex is similar to that of free HRP, but conversion to the intermediate- and low-spin states is complete at a higher temperature than in the free enzyme. Addition of benzohydroxamic acid (BHA) to HRP shifts the RR frequencies to those corresponding to a 6-coordinate high-spin species at both room and low temperature. Two upsilon (C = C) stretching modes are observed for HRP and its donor complexes, indicating that the vinyl groups are inequivalent. On BHA binding, one of the vinyl modes and upsilon 37 (Eu) are enhanced, suggesting symmetry lowering of the heme site.     

A secondary xylan-binding site enhances the catalytic activity of a single-domain family 11 glycoside hydrolase

Bacillus circulans xylanase (BcX) is a single-domain family 11 glycoside hydrolase. Using NMR-monitored titrations, we discovered that an inactive variant of this enzyme, E78Q-BcX, bound xylooligosaccharides not only within its pronounced active site (AS) cleft, but also at a distal surface region. Chemical shift perturbation mapping and affinity electrophoresis, combined with mutational studies, identified the xylan-specific secondary binding site (SBS) as a shallow groove lined by Asn, Ser, and Thr residues and with a Trp at one end. The AS and SBS bound short xylooligosaccharides with similar dissociation constants in the millimolar range. However, the on and off-rates to the SBS were at least tenfold faster than those of kon approximately 3x10(5) M(-1) s(-1) and koff approximately 1000 s(-1) measured for xylotetraose to the AS of E78Q-BcX. Consistent with their structural differences, this suggests that a conformational change in the enzyme and/or the substrate is required for association to and dissociation from the deep AS, but not the shallow SBS. In contrast to the independent binding of small xylooligosaccharides, high-affinity binding of soluble and insoluble xylan, as well as xylododecaose, occurred cooperatively to the two sites. This was evidenced by an approximately 100-fold increase in relative Kd values for these ligands upon mutation of the SBS. The SBS also enhances the activity of BcX towards soluble and insoluble xylan through a significant reduction in the Michaelis KM values for these polymeric substrates. This study provides an unexpected example of how a single domain family 11 xylanase overcomes the lack of a carbohydrate-binding module through the use of a secondary binding site to enhance substrate specificity and affinity.     

Binding kinetics of engineered mutants provide insight about the pathway for entering and exiting the intestinal fatty acid binding protein

To better understand the mechanism by which fatty acids bind to and dissociate from the binding cavities of fatty acid binding proteins (FABPs), we constructed 31 single amino acid mutants of the intestinal FABP (I-FABP) and determined the rate constants for binding and dissociation, primarily for long-chain fatty acids (FA). FA dissociation from these proteins was measured both by the ADIFAB method and by the change in tryptophan fluorescence of the FABPs. Rate constants for binding (kon) were calculated from the rate constants for dissociation (koff) and the equilibrium binding affinities. Amino acid substitutions were made at locations within the binding cavity, in the region of the gap between the betaD- and betaE-strands, and within the "portal" region of the protein. The koff values for the mutant proteins ranged from about 20-fold slower to 4-fold faster than the wild-type (WT) protein. Values for kon were as much as 20-fold slower than the WT protein, but in no case was kon significantly faster than the WT. Mutants with slower and faster koff values were generally those involving sites within the binding cavity and, relative to the WT protein, revealed higher and lower affinities, respectively. Reduced rates of binding were generally, but not exclusively, associated with sites within the portal region. For example, for F68A which is located closer to the opposite end of the protein from the portal region, the kon is more than 10-fold slower than WT. Even for these distal sites, however, the evidence is consistent with reductions in kon being due to alterations of the portal region. Binding affinities and rate constants measured as a function of ionic strength also suggest that the FA initially binds, through an electrostatic interaction, to Arg-56 on the surface of the protein, before inserting into the binding cavity. Thus, the results of this study are consistent with FA binding to I-FABP involving an initial interaction with Arg-56 followed by insertion of the FA, through the portal region, into the binding cavity and with a reversal of these steps for the dissociation reaction.     

17O-water and cyanide ligation by the active site iron of protocatechuate 3,4-dioxygenase. Evidence for displaceable ligands in the native enzyme and in complexes with inhibitors or transition state analogs

Hyperfine broadening is observable in the EPR spectrum of Brevibacterium fuscum protocatechuate 3,4-dioxygenase after lyophilization and rehydration in 17O-enriched water, demonstrating H2O ligation to the active site iron. Lack of detectable broadening in the sharp features of the spectra of three substrate complexes suggests that H2O is displaced by substrate. Water is bound in the monodentate complex with the competitive inhibitor 3-hydroxybenzoate which binds directly to the iron showing that two iron ligation sites can be occupied by nonprotein ligands. Ketonized substrate analogs which mimic a proposed transition state of the reaction cycle, 2-hydroxyisonicotinic acid N-oxide (2-OHINO) and 6-hydroxynicotinic acid N-oxide (6-OH NNO), have H2O bound in their final, bleached enzyme complexes, suggesting that these complexes are also monodentate. In contrast, a transient, initial complex of 6-OH NNO which is spectrally similar to the substrate complex, apparently does not have H2O bound. Cyanide binding occurs in two steps. The active site Fe3+ of the initial, rapidly formed, violet complex is high spin while that of the second, slowly formed, green complex is low spin; a unique state for mononuclear non-heme iron enzymes. The data suggest that the Fe-CN- and Fe-(CN-)2 complexes form sequentially. CN- binds to enzyme complexes with 2-OH INO and 6-OH NNO in one step to yield high spin Fe3+ species. In contrast, preformed substrate complexes prevent CN- binding. CN- binding eliminates the broadening due to 17O-water in the EPR spectra of both native enzyme and the enzyme-ketonized analog complexes. A model is proposed in which H2O is displaced by bidentate binding of the substrate but can potentially rebind after a subsequent substrate ketonization. The proximity of the vacatable H2O-binding site of the iron to the site of oxygen insertion suggests, however, that this site may serve to stabilize an oxygenated intermediate during the reaction cycle.     

Substrates modulate the rate-determining step for CO binding in cytochrome P450cam (CYP101). A high-pressure stopped-flow study.

The high-pressure stopped-flow technique is applied to study the CO binding in cytochrome P450cam (P450cam) bound with homologous substrates (1R-camphor, camphane, norcamphor and norbornane) and in the substrate-free protein. The activation volume DeltaV # of the CO on-rate is positive for P450cam bound with substrates that do not contain methyl groups. The kon rate constant for these substrate complexes is in the order of 3 x 10(6) M(-1) x s(-1). In contrast, P450cam complexed with substrates carrying methyl groups show a negative activation volume and a low kon rate constant of approximately 3 x 10(4) M(-1) x s(-1). By relating kon and DeltaV # with values for the compressibility and the influx rate of water for the heme pocket of the substrate complexes it is concluded that the positive activation volume is indicative for a loosely bound substrate that guarantees a high solvent accessibility for the heme pocket and a very compressible active site. In addition, subconformers have been found for the substrate-free and camphane-bound protein which show different CO binding kinetics.     

Resonance Raman studies of sterically hindered cyanomet "strapped" hemes. Effects of ligand distortion and base tension on iron-carbon bond

We report resonance Raman studies of the iron-carbon bond stretching vibrations, nu(Fe-CN), in sterically hindered and unhindered heme (FeIII)-CN- complexes. The sterically hindred "strapped hemes" are equipped with a covalently linked 13-, 14-, or 15-atom hydrocarbon chain across one face of the heme; these are called FeSP-13, FeSP-14, and FeSP-15, respectively. These straps would presumably exert a sideway shearing strain to force the linear ligands (e.g., CN- and CO) to be tilted and/or bent. The shorter the chain length, the weaker the ligand binding affinity because of a greater steric hindrance. This study reveals that the nu(Fe-CN) frequency decreases as the chain length is decreased, in contrast with the CO complexes, where the nu(Fe-CO) frequency increases as the chain length is decreased. For the heme-CN- complexes (with N-methylimidazole as a base), the nu(Fe-CN) frequencies are: heme 5 (unhindered), 451 cm-1; FeSP-15, 447 cm-1; FeSP-14, 447 cm-1; FeSP-13, 445 cm-1. For the heme-CO complexes (with N-methylimidazole as a base), the nu(Fe-CO) frequencies are: heme 5, 495 cm-1; FeSP-15, 509 cm-1; FeSP-14, 512 cm-1; FeSP-13, 514 cm-1 (Yu, N.-T., E. A. Kerr, B. Ward, and C. K. Chang, 1983, Biochemistry, 22:4534-4540). We have also studied the cyanide complexes with three different bases (pyridine, N-methylimidazole and 1,2-dimethylimidazole), and found that the trans-effect of cyanide complex is different from that of CO complexes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of aromatic donor molecules with manganese(III) reconstituted horseradish peroxidase: proton nuclear magnetic resonance and optical difference spectroscopic studies

The interaction of aromatic donor molecules with manganese(III) protoporphyrin-apohorseradish peroxidase complex [Mn(III)HRP] was investigated by optical difference spectroscopy and relaxation rate measurements of 1H resonances of aromatic donor molecules (at 500 MHz). pH dependence of substrate proton resonance line-widths indicated that the binding was facilitated by protonation of an amino acid residue (with a pKa of 6.1), which is presumably distal histidine. Dissociation constants were evaluated from both optical difference spectroscopy and 1H-NMR relaxation measurements (pH 6.1). The dissociation constants of aromatic donor molecules were not affected by the presence of excess of I-, CN- and SCN-. From competitive binding studies it was shown that all these aromatic donor molecules bind to Mn(III)HRP at the same site, which is different from the binding site of I-, CN- and SCN-. Comparison of the dissociation constants between the different substrates suggests that hydrogen bonding of the donors with distal histidyl amino acid and hydrophobic interaction between the donors and active site contribute significantly towards the associating forces. Free energy, entropy and enthalpy changes associated with the Mn(III)HRP-substrate equilibrium have been evaluated. These thermodynamic parameters were found to be all negative. Distances of the substrate protons from the paramagnetic manganese ion of Mn(III)HRP were found to be in the range of 7.7 to 9.4 A. The Kd values, the thermodynamic parameters and the distances of the bound aromatic donor protons from metal center in the case of Mn(III)HRP were found to be very similar as in the case of native Fe(III)HRP.     

Differences in the binding of aromatic substrates to horseradish peroxidase revealed by fluorescence line narrowing

The heme in horseradish peroxidase (HRP) isoenzyme C was replaced by mesoporphyrin (MP), and the binding effect of the aromatic substrates benzo-and naphthohydroxamic acid (BHA, NHA), resorcinol (RE), isomeric resorcylic acids (alpha-, beta-, gamma-RE), and hydroquinone (HQ) was studied at pH 5 by conventional and laser-excited fluorescence spectroscopy on the basis of the signal of the porphyrin. Under laser excitation at cryogenic temperatures site selection was demonstrated, and the fluorescence line narrowing data were used to characterize the HRP/substrate complexes by the inhomogeneous distribution function for the S0----S1 (0----0) transition energy and the vibrational energies in the S1 electronic state. A comparison with ground-state vibrational energies for MP in chloroform/ether showed a downward shift in vibrational energies for S1 by approximately 20 cm-1. The association characteristics of the substrates were in accordance with previous literature data indicating NHA to be of the strongest binding affinity. For BHA, spectral evidence was obtained for a second type of binding site where hydrophobic interactions with the porphyrin ring may be possible. The effect of the RE's was similar to each other, but only beta-RE showed saturation. Complexation in every case caused the strong reduction of the splitting in the 0----0 transition energy for the tautomeric forms of MP and an increase in the 0----0 energy by 100-200 cm-1 depending on the substrate. The substrate binding also affected the phonon coupling of vibronic transitions exciting into the delta v = 927- and 976-cm-1 modes; in the latter case, the vibrational energy was also increased to 983 cm-1 for beta-RE. In the same energy range, however, the transition into the delta nu = 958-960-cm-1 mode was not affected by binding. Both the magnitude of the energy shifting and the change in the strength of phonon coupling gave the same relation, BHA less than NHA less than HQ less than RE's, indicating a common conformational origin. A reduction of the fluctuational freedom of the protein chain at room temperature within the heme pocket was suggested on the basis of the reduction of the width of the inhomogeneous distribution of 0----0 energies (from 60-70 to approximately 30 cm-1 in case of HRP/HQ) upon substrate binding. Ways to relate the transition energy splitting and shifting effects to conformational changes are discussed by invoking the Jahn-Teller effect.     

Infrared absorption study of the heme pocket dynamics of carbonmonoxyheme proteins

The temperature dependencies of the infrared absorption CO bands of carboxy complexes of horseradish peroxidase (HRP(CO)) in glycerol/water mixture at pH 6.0 and 9.3 are interpreted using the theory of optical absorption bandshape. The bands' anharmonic behavior is explained assuming that there is a higher-energy set of conformational substates (CSS(h)), which are populated upon heating and correspond to the protein substates with disordered water molecules in the heme pocket. Analysis of the second moments of the CO bands of the carboxy complexes of myoglobin (Mb(CO)) and hemoglobin (Hb(CO)), and of HRP(CO) with benzohydroxamic acid (HRP(CO)+BHA), shows that the low energy CSS(h) exists also in the open conformation of Mb(CO), where the heme pocket is spacious enough to accommodate a water molecule. In the HRP(CO)+BHA and closed conformations of Mb(CO) and Hb(CO), the heme pocket is packed with BHA and different amino acids, the CSS(h) has much higher energy and is hardly populated even at the highest temperatures. Therefore only motions of these amino acids contribute to the band broadening. These motions are linked to the protein surface and frozen in the glassy matrix, whereas in the liquid solvent they are harmonic. Thus the second moment of the CO band is temperature-independent in glass and is proportional to the temperature in liquid. The temperature dependence of the second moment of the CO peak of HRP(CO) in the trehalose glass exhibits linear coupling to an oscillator. This oscillator can be a moving water molecule locked in the heme pocket in the whole interval of temperatures or a trehalose molecule located in the heme pocket.     

Effect of enzyme and ligand protonation on the binding of folates to recombinant human dihydrofolate reductase: implications for the evolution of eukaryotic enzyme efficiency.

There is marked pH dependence of the rate constant (koff) for tetrahydrofolate (H4folate) dissociation from its ternary complex with human dihydrofolate reductase (hDHFR) and NADPH. Similar pH dependence of H4folate dissociation from the ternary complex of a variant of hDHFR with the substitution Phe31----Leu (F31L hDHFR) causes this dissociation to become rate limiting in the enzyme mechanism at pH approximately 5, and this accounts for the marked decrease in kcat for this variant as the pH is decreased from 7 to 5. This decreased kcat at low pH is not seen for most DHFRs. koff for dissociation of folate, dihydrofolate (H2folate), and H4folate from their binary complexes with hDHFR is similarly pH dependent. For all the complexes examined, the pH dependence of koff in the range pH 5-7 is well described by a pKa of about 6.2 and must be due to ionization of a group on the enzyme. In the higher pH range (7-10), koff increases further as the pH is raised, and this relation is governed by a second pKa which is close to the pKa for ionization of the amide group (HN3-C4O) of the respective ligands. Thus, ionization of the ligand amide group also increases koff. Evidence is presented that the dependence of pH on koff for hDHFR accounts for the shape of the kcat versus pH curve for both hDHFR as well as its F31L variant and contributes to the higher efficiency of hDHFR compared with bacterial DHFR.     

pH dependence of the inhibition of chymotrypsin by a peptidyl trifluoromethyl ketone

The effects of pH on the kinetics of association and dissociation of chymotrypsin and the dipeptidyl trifluoromethyl ketone (TFK) N-acetyl-L-leucyl-L-phenylalanyltrifluoromethane (1) were examined through the pH range 4-9.5. The pH dependence of the association rate (kon) is similar to that of kcat/Km for ester and peptide substrates and is dependent on two pK's at 7.0 and 8.9. We assign these pK's to the active site His and to the amino group of the N-terminal isoleucine residue. Ki for the complex of 1 and chymotrypsin has a pH dependence very similar to that of kon, and we conclude that the same ionizable groups which determine the pH dependence of kon are involved. The dissociation constant of the enzyme-inhibitor complex (koff) shows no pH dependence between pH 4 and pH 9.5. The data indicate that the inhibitor reacts with a form of the enzyme in which His 57 is unprotonated, and the resulting complex contains no groups which ionize between pH 4 and pH 9.5. This is consistent with conclusions previously reached from NMR data (Liang & Abeles, 1987). These experiments led to the conclusion that 1 reacts with chymotrypsin to form a tetrahedral complex in which His 57 is protonated (pK greater than 9.5) and the OH group of serine 195 has added to the carbonyl group of 1 to form an ionized hemiketal (pK less than 4.9). The pK of His 57 is increased by greater than 3 units over that in the free enzyme, and the pK of the hemiketal decreased by greater than 4 units compared to the pK in solution.(ABSTRACT TRUNCATED AT 250 WORDS)