Asp129 of low molecular weight protein tyrosine phosphatase is involved in leaving group protonation

Site-directed mutagenesis was used to explore the functions of a number of acidic residues of bovine low molecular weight protein tyrosine phosphatase. Residues Asp-129, Asp-56, and Asp-92 were mutated to Ala or Asn. The mutant enzymes D56A, D56N, and D92A showed no significant changes in Vmax values, although they did exhibit significantly altered Km values. In contrast, the D129A mutant enzyme exhibited a greater than 2000-fold reduction in Vmax, using p-nitrophenyl phosphate as a substrate. The Vmax values of D129A also exhibited a leaving group dependence, an altered solvent isotope effect of VmaxH/VmaxD of 0.78, and a lack of dependence on the presence of alternative phosphate acceptor alcohols, all properties that distinguish this mutant from wild type enzyme. The differences are due to a change of the rate-limiting step of the catalytic reaction. Asp-129 is concluded to be the proton donor to the leaving group in the phosphorylation step, and its mutation to alanine results in a reduced Vmax value and a change in the rate-limiting step of the catalysis from dephosphorylation to phosphorylation. Mechanistic considerations suggest that other phosphotyrosyl phosphatases having cysteine at the active site may be expected to have a similar requirement for a proton donor.     

Oxidation kinetics of ethanol by human cytochrome P450 2E1. Rate-limiting product release accounts for effects of isotopic hydrogen substitution and cytochrome b5 on steady-state kinetics

A number of cytochrome P450 (P450) 2E1 substrates are known to show kinetic deuterium isotope effects of approximately 5 on Km (DK = DKm/HKm), but not on kcat, in rat liver microsomes (e.g. N-nitrosodimethylamine, ethanol, and CH2Cl2). We observed DKm values of 3-5 for recombinant human P450 2E1-catalyzed ethanol oxidation. Replacing NADPH and O2 with the oxygen surrogate cumene hydroperoxide yielded similar results. Ferric P450 2E1 reduction was fast (k >1000 min-1) even in the absence of substrate. These results indicate that the basis for the increase in Km is in the latter portion of the catalytic cycle. The intrinsic isotope effect (Dk) for ethanol oxidation was determined (competitively) to be 3.8, indicating that C-H bond cleavage is isotopically sensitive. Pre-steady-state studies showed a burst of product formation (k = 410 min-1), with the burst amplitude corresponding to the P450 concentration. Deuteration of ethanol resulted in an isotope effect of 3.2 on the rate of the burst. We conclude that product release is rate-limiting in the oxidation of ethanol to acetaldehyde by P450 2E1. The steady-state kinetics can be described by a paradigm in which the kcat approximates the rate of product release, and Km is an expression in which the denominator is dominated by the rate of C-H bond breaking.     

Improved binding of cytochrome P450cam substrate analogues designed to fill extra space in the substrate binding pocket

Cytochrome P450cam catalyzes the 5-exo-hydroxylation of camphor. Camphor analogues were designed to fill an empty region of the substrate binding pocket with the expectation that they would bind more tightly than camphor itself due to increased van der Waals interactions with the protein and the displacement of any solvent occupying this site. A series of compounds (endo-borneol methyl ether, endo-borneol propyl ether, endo-borneol allyl ether and endo-borneol dimethyl allyl ether) were synthesized with substituents at the camphor carbonyl oxygen. The spin conversion and thermodynamic properties of this series of compounds were measured for wild type and Y96F mutant cytochrome P450cam and were interpreted in the context of molecular dynamics simulations of the camphor analogues in the P450 binding site and in solution. Compounds with a 3-carbon chain substituent were predicted to match the size of the unoccupied region most optimally and thus bind best. Consistent with this prediction, the borneol allyl ether binds to cytochrome P450cam with highest affinity with a Kd = 0.6 +/- 0.1 microM (compared to a Kd = 1.7 +/- 0.2 microM for camphor under the same experimental conditions). Binding of the camphor analogues to the Y96F mutant is much enhanced over the binding of camphor, indicating that hydrogen bonding plays a less important role in binding of these analogues. Binding enthalpies calculated from the simulations, taking all solvent contributions into account, agree very well with experimental binding enthalpies. Binding affinity is not however correlated with the calculated binding enthalpy because the binding of the substrate analogues is characterized by enthalpy-entropy compensation. The new compounds are useful probes for further studies of the mechanism of cytochrome P450cam due to their high binding affinities and high spin properties.     

Contribution to activity of histidine-aromatic, amide-aromatic, and aromatic-aromatic interactions in the extended catalytic site of cysteine proteinases

Within the papain family of cysteine proteinases few other residues in addition to the catalytic triad, Cys25-His159-Asn175 (papain numbering) are completely conserved [Berti & Storer (1995) J. Mol. Biol. 246, 273-283]. One such residue is tryptophan 177 which participates in a Trp-His-type interaction with the catalytic His159. In all enzymes of this class for which a three-dimensional structure has been reported, an additional highly conserved tryptophan, Trp181, also interacts with Trp177 via an aromatic-aromatic interaction in which the planes of the indole rings are essentially perpendicular. Also, both indole rings participate as pseudo-hydrogen bond acceptors in interactions with the two side chain amide protons of Asn175. Clearly, the proximity of Trp177 and Trp181 to the catalytic triad residues His159 and Asn175 and their network of interactions points to potential contributions of these aromatic residues to catalysis. In this paper, using cathepsin S, a naturally occurring variant that has a phenylalanine residue at position 181, we report the kinetic characterization of mutants of residues 175, 177, and 181. The results are interpreted in terms of the side chain contributions to catalytic activity and thiolate-imidazolium ion-pair stability. For example, the side chain of Asn175 has a major influence on the ion-pair stability presumably through its hydrogen bond to His159. The magnitude of this effect is modulated by Trp177, which shields the His159-Asn175 hydrogen bond from solvent. The His159-Trp177 interaction also contributes significantly to ion-pair stability; however, Trp181 and its interactions with Asn175 and Trp177 do not influence ion-pair stability to a significant degree. The observation that certain mutations at positions 177 and 181 result in a reduction of kcat/Km but do not appear to influence ion-pair stability probably reflects the contributions of these residues to substrate binding.     

Probing the mechanism of proton coupled electron transfer to dioxygen: the oxidative half-reaction of bovine serum amine oxidase

Bovine serum amine oxidase (BSAO) catalyzes the oxidative deamination of primary amines, concomitant with the reduction of molecular oxygen to hydrogen peroxide via a ping-pong mechanism. A protocol has been developed for an analysis of chemical and kinetic mechanisms in the conversion of dioxygen to hydrogen peroxide. Steady-state kinetics show that two groups need to be deprotonated to facilitate the oxidative half-reaction. The pH dependence of Vmax/Km(O2) reveals pKa's of 6.2 +/- 0.3 and 7.0 +/- 0.2, respectively. A pKa of 7.2 +/- 0.1 has been obtained from a titration of anaerobically reduced BSAO using UV-vis spectrophotometry. The near identity of the pKa obtained from the reduced enzyme titration with the second pKa from steady-state kinetics suggests that this second pKa arises from the reduced cofactor. The assignment of pKa is supported by the observed pH dependence for formation of the cofactor semiquinone signal, detected by EPR spectroscopy under anaerobic conditions. To address the nature of rate-limiting steps in the oxidative half-reaction, the solvent isotope effect, viscosity effect, and O-18 isotope effect on Vmax/Km(O2) have been determined. The solvent isotope effect is indistinguishable from unity, ruling out a proton transfer as a rate-determining step. Use of glucose as a solvent viscosogen shows no viscosity effect, indicating that binding of oxygen is not in the rate-determining step. The O-18 kinetic isotope effect is independent of pH with an average value of 18(V/K) = 1.0097 +/- 0. 0010. This has been compared to calculated equilibrium O-18 isotope effects for various dioxygen intermediate species [Tian and Klinman (1993) J. Am. Chem. Soc. 115, 8891], leading to the conclusion that either the first electron transfer to dioxygen or the desorption of product peroxide from a Cu(II)-OOH complex could be the rate-limiting step. The distribution of steady-state enzyme species was, therefore, analyzed through a combination of stopped-flow experiments and analysis of DV and D(V/K) for benzylamine oxidation. We conclude that the major species accumulating in the steady state are the oxidized cofactor-substrate Schiff base complex and the reduced, aminoquinol form of cofactor. These data rule out a slow release of product hydroperoxide from the aminoquinone form of enzyme, leading to the conclusion that the first electron transfer from substrate-reduced cofactor to dioxygen is the rate-determining step in the oxidative half-reaction. This step is also estimated to be 40% rate-limiting in kcat. An important mechanistic conclusion from this study is that dioxygen binding is a separate step from the rate-limiting electron-transfer step to form superoxide. On the basis of a recently determined X-ray structure for the active form of a yeast amine oxidase from Hansenula polymorpha [Li et al. (1998) Structure 6, 293], a hydrophobic space has been identified near the O-2 position of reduced cofactor as the putative dioxygen binding site. Movement of superoxide from this site onto the Cu(II) at the active site may occur prior to further electron transfer from cofactor to superoxide.     

Probing the mechanism of inosine monophosphate dehydrogenase with kinetic isotope effects and NMR determination of the hydride transfer stereospecificity

The mechanism of human type II inosine monophosphate dehydrogenase has been probed by measurements of primary deuterium kinetic isotope effects, and by determination of the stereochemical course of the reaction. The deuterium isotope effects on Vmax from [2-deutero]-IMP are unity for reactions with a variety of monovalent cation activators (K+, NH4+, Na+, Rb+) of various efficacy. In each case normal effects on Vmax/K(m) in the range of 1.9 to 3.5 are observed for both IMP and NAD, and are larger for NAD. These results demonstrate that both substrates can dissociate from the E.M+.IMP.NAD complex, therefore the kinetic mechanism is not ordered as previous steady-state kinetic studies have suggested. Comparison of reaction rates in D2O and H2O show no 2H isotope effect on Vmax, and a < or = twofold decrease in Vmax/K(m); thus, a proton transfer from solvent is not rate-limiting in turnover. The NMR spectrum of the [4-deutero]NADH produced in the reaction of [2-deutero]-IMP and NAD shows that the hydrogen is transferred to the B, or pro-S, side of the nicotinamide ring. Presteady-state kinetic experiments reveal a burst of NADH formation in the first turnover, demonstrating that a late step in the mechanism is rate-limiting. The rate of the burst phase is reduced approximately twofold with [2-deutero]IMP as substrate, indicating that the hydride transfer step is kinetically significant early in the reaction.     

Hydrogen bond network in the distal site of peroxidases: spectroscopic properties of Asn70 --> Asp horseradish peroxidase mutant

The distal His in peroxidases forms a hydrogen bond with the adjacent Asn, which is highly conserved among many plant and fungal peroxidases. Our previous work [Nagano, S., Tanaka, M., Ishimori, K., Watanabe, Y., & Morishima, I. (1996) Biochemistry 35, 14251-14258] has revealed that the replacement of Asn70 in horseradish peroxidase C (HRP) by Val (N70V) and Asp (N70D) discourages the oxidation activity for guaiacol, and the elementary reaction rate constants for the mutants was decreased by 10-15-fold. In order to delineate the structure-function relationship of the His-Asn couple in peroxidase activity, heme environmental structures of the HRP mutant, N70D, were investigated by CD, 1H NMR, and IR spectroscopies as well as Fe2+/Fe3+ redox potential measurements. While N70D mutant exhibited quite similar CD spectra and redox potential to those of native enzyme, the paramagnetic NMR spectrum clearly showed that the hydrogen bond between the distal His and Asp70 is not formed in the mutant. The disappearance of the splitting in the 1H NMR signal of heme peripheral 8-methyl group observed in 50% H2O/50% D2O solution of N70D-CN suggests that the hydrogen bond between the distal His and heme-bound cyanide is also disrupted by the mutation, which was supported by the low C-N vibration frequency and large dissociation constant of the heme-bound cyanide in the mutant. Together with the results from various spectroscopies and redox potentials, we can conclude that the improper positioning of the distal His induced the cleavages of the hydrogen bonds around the distal His, resulting in the substantial decrease of the catalytic activity without large structural alterations of the enzyme. The His-Asn hydrogen bond in the distal site of peroxidases, therefore, is essential for the catalytic activity by controlling the precise location of the distal His.     

Contribution of cutinase serine 42 side chain to the stabilization of the oxyanion transition state

Cutinase from the fungus Fusarium solani pisi is a lipolytic enzyme able to hydrolyze both aggregated and soluble substrates. It therefore provides a powerful tool for probing the mechanisms underlying lipid hydrolysis. Lipolytic enzymes have a catalytic machinery similar to those present in serine proteinases. It is characterized by the triad Ser, His, and Asp (Glu) residues, by an oxyanion binding site that stabilizes the transition state via hydrogen bonds with two main chain amide groups, and possibly by other determinants. It has been suggested on the basis of a covalently bond inhibitor that the cutinase oxyanion hole may consist not only of two main chain amide groups but also of the Ser42 O gamma side chain. Among the esterases and the serine and the cysteine proteases, only Streptomyces scabies esterase, subtilisin, and papain, respectively, have a side chain residue which is involved in the oxyanion hole formation. The position of the cutinase Ser42 side chain is structurally conserved in Rhizomucor miehei lipase with Ser82 O gamma, in Rhizopus delemar lipase with Thr83 O gamma 1, and in Candida antartica B lipase with Thr40 O gamma 1. To evaluate the increase in the tetrahedral intermediate stability provided by Ser42 O gamma, we mutated Ser42 into Ala. Furthermore, since the proper orientation of Ser42 O gamma is directed by Asn84, we mutated Asn84 into Ala, Leu, Asp, and Trp, respectively, to investigate the contribution of this indirect interaction to the stabilization of the oxyanion hole. The S42A mutation resulted in a drastic decrease in the activity (450-fold) without significantly perturbing the three-dimensional structure. The N84A and N84L mutations had milder kinetic effects and did not disrupt the structure of the active site, whereas the N84W and N84D mutations abolished the enzymatic activity due to drastic steric and electrostatic effects, respectively.     

Evaluation of the role of His447 in the reaction catalyzed by cholesterol oxidase

Cholesterol oxidase catalyzes the oxidation and isomerization of cholesterol to cholest-4-en-3-one via cholest-5-en-3-one. It has been proposed that His447 acts as the general base catalyst for oxidation, and that the resulting imidazolium ion formed acts as an electrophile for isomerization. In this work, we undertook an assessment of the proposed dual roles of His447 in the oxidation and isomerization reactions. To test its role, we constructed five mutants, H447Q, H447N, H447E, H447D, and H447K, that introduce hydrogen bond donors and acceptors and carboxylate bases at this position, and a sixth mutant, E361Q, to test the interplay between His447 and Glu361. These mutants were characterized using steady-state kinetics and deuterium substrate and solvent isotope effects. For those mutants that catalyze either oxidation of cholesterol or isomerization of cholest-5-en-3-one, the Km's vary no more than 3-fold relative to wild type. H447K is inactive in both oxidation (> 100,000-fold reduced) and isomerization assays (> 10,000-fold reduced). H447E and H447D do not catalyze oxidation (> 100,000-fold reduced), but do catalyze isomerization, 10(4) times slower than wild type. The k(cat) for H447Q is 120-fold lower than wild type for oxidation, and the same as wild type for isomerization. The k(cat) for H447N is 4400-fold lower than wild type for oxidation, and is 30-fold lower than wild type for isomerization. E361Q does not catalyze isomerization (> 10,000-fold reduced), and the k(cat) for oxidation is 30-fold lower than wild type. The substrate deuterium kinetic isotope effects for the wild-type and mutant-catalyzed oxidation reactions suggest that mutation of His447 to an amide results in a change of the rate-determining step from hydride transfer to hydroxyl deprotonation. The deuterium solvent and substrate kinetic isotope effects for isomerization indicate that an amide at position 447 is an effective electrophile to catalyze formation of a dienolic intermediate. Moreover, consideration of kinetic and structural results together suggests that a hydrogen bonding network involving His447, Glu361 and Asn485, Wat541, and substrate serves to position the substrate and coordinate general base and electrophilic catalysis. That is, in addition to its previously demonstrated role as base for deprotonation of carbon-4 during isomerization, Glu361 has a structural role and may act as a general base during oxidation. The His447, Asn485, Glu361, and Wat541 residues are conserved in other GMC oxidoreductases. Observation of this catalytic tetrad in flavoproteins of unknown function may be diagnostic for an ability to oxidize unactivated alcohols.     

High-resolution crystal structures of delta5-3-ketosteroid isomerase with and without a reaction intermediate analogue

Bacterial Delta5-3-ketosteroid isomerase (KSI) catalyzes a stereospecific isomerization of steroid substrates at an extremely fast rate, overcoming a large disparity of pKa values between a catalytic residue and its target. The crystal structures of KSI from Pseudomonas putida and of the enzyme in complex with equilenin, an analogue of the reaction intermediate, have been determined at 1.9 and 2.5 A resolution, respectively. The structures reveal that the side chains of Tyr14 and Asp99 (a newly identified catalytic residue) form hydrogen bonds directly with the oxyanion of the bound inhibitor in a completely apolar milieu of the active site. No water molecule is found at the active site, and the access of bulk solvent is blocked by a layer of apolar residues. Asp99 is surrounded by six apolar residues, and consequently, its pKa appears to be elevated as high as 9.5 to be consistent with early studies. No interaction was found between the bound inhibitor and the residue 101 (phenylalanine in Pseudomonas testosteroni and methionine in P. putida KSI) which was suggested to contribute significantly to the rate enhancement based on mutational analysis. This observation excludes the residue 101 as a potential catalytic residue and requires that the rate enhancement should be explained solely by Tyr14 and Asp99. Kinetic analyses of Y14F and D99L mutant enzymes demonstrate that Tyr14 contributes much more significantly to the rate enhancement than Asp99. Previous studies and the structural analysis strongly suggest that the low-barrier hydrogen bond of Tyr14 (>7.1 kcal/mol), along with a moderate strength hydrogen bond of Asp99 ( approximately 4 kcal/mol), accounts for the required energy of 11 kcal/mol for the transition-state stabilization.     

His...Asp catalytic dyad of ribonuclease A: structure and function of the wild-type, D121N, and D121A enzymes

The side chains of histidine and aspartate residues form a hydrogen bond in the active sites of many enzymes. In serine proteases, the His...Asp hydrogen bond of the catalytic triad is known to contribute greatly to catalysis, perhaps via the formation of a low-barrier hydrogen bond. In bovine pancreatic ribonuclease A (RNase A), the His...Asp dyad is composed of His119 and Asp121. Previously, site-directed mutagenesis was used to show that His119 has a fundamental role, to act as an acid during catalysis of RNA cleavage [Thompson, J. E., and Raines, R. T. (1994) J. Am. Chem. Soc. 116, 5467-5468]. Here, Asp121 was replaced with an asparagine or alanine residue. The crystalline structures of the two variants were determined by X-ray diffraction analysis to a resolution of 1.6 A with an R-factor of 0.18. Replacing Asp121 with an asparagine or alanine residue does not perturb the overall conformation of the enzyme. In the structure of D121N RNase A, Ndelta rather than Odelta of Asn121 faces His119. This alignment in the crystalline state is unlikely to exist in solution because catalysis by the D121N variant is not compromised severely. The steady-state kinetic parameters for catalysis by the wild-type and variant enzymes were determined for the cleavage of uridylyl(3'-->5')adenosine and poly(cytidylic acid), and for the hydrolysis of uridine 2',3'-cyclic phosphate. Replacing Asp121 decreases the values of kcat/Km and kcat for cleavage by 10-fold (D121N) and 10(2)-fold (D121A). Replacing Asp121 also decreases the values of kcat/Km and kcat for hydrolysis by 10(0. 5)-fold (D121N) and 10-fold (D121A) but has no other effect on the pH-rate profiles for hydrolysis. There is no evidence for the formation of a low-barrier hydrogen bond between His119 and either an aspartate or an asparagine residue at position 121. Apparently, the major role of Asp121 is to orient the proper tautomer of His119 for catalysis. Thus, the mere presence of a His...Asp dyad in an enzymic active site is not a mandate for its being crucial in effecting catalysis.     

Evidence for a functional role of the dynamics of glycine-121 of Escherichia coli dihydrofolate reductase obtained from kinetic analysis of a site-directed mutant

Two-dimensional heteronuclear (1H-15N) nuclear magnetic relaxation studies of dihydrofolate reductase (DHFR) from Escherichia coli have demonstrated that glycine-121 which is 19 A from the catalytic center of the enzyme has large-amplitude backbone motions on the nanosecond time scale [Epstein, D. M., Benkovic, S. J., and Wright, P. E. (1995) Biochemistry 34, 11037-11048]. In order to probe the dynamic-function relationships of this residue, we constructed a mutant enzyme in which this glycine was changed to valine. Equilibrium binding studies indicated that the Val-121 mutant retained wild-type binding properties with respect to dihydrofolate and tetrahydrofolate; however, binding to NADPH and NADP+ was decreased by 40-fold and 2-fold, respectively, relative to wild-type DHFR. Single-turnover experiments indicated that hydride transfer was reduced by 200-fold to a rate of 1.3 s-1 and was the rate-limiting step in the steady state. Interestingly, pre-steady-state kinetic analysis of the Val-121 mutant revealed a conformational change which preceded chemistry that occurred at a rate of 3.5 s-1. If this step exists in the kinetic mechanism of the wild-type enzyme, then it would be predicted to occur at a rate of approximately 2000 s-1. Glycine-121 was also changed to alanine, serine, leucine, and proline. While the Ala-121 and Ser-121 mutants behaved similar to wild-type DHFR, the Leu-121 and Pro-121 mutants behaved like Val-121 DHFR in that hydride transfer was the rate-limiting step in the steady state and a conformational change preceding chemistry was observed. Finally, insertion of a glycine or valine between amino acids 121 and 122 produced mutant enzymes with properties similar to wild-type or Val-121 DHFRs, respectively. Taken together, these results provide compelling evidence for dynamic coupling of a remote residue to kinetic events at the active site of DHFR.     

A source of response regulator autophosphatase activity: the critical role of a residue adjacent to the Spo0F autophosphorylation active site

Two-component signaling systems are used by bacteria, plants, and lower eukaryotes to adapt to environmental changes. The first component, a protein kinase, responds to a signal by phosphorylating the second component; a response regulator protein that often acts by inducing the expression of specific genes. Response regulators also have an autophosphatase activity that ensures that the proteins are not permanently activated by phosphorylation. The magnitude of this activity varies by at least 1000-fold between various response regulators, and the molecular features responsible for this varied autophosphatase activity have not been clearly defined. Using wild-type and mutant derivatives of the sporulation response regulator Spo0F, it has been demonstrated that a key residue in determining the magnitude of this activity is that at position 56 of Spo0F approximately P; this residue is adjacent to the site of phosphorylation, Asp 54. For example, Spo0F approximately P K56N has a 23-fold greater autophosphatase activity (t1/2 = 8 min) than wild-type Spo0F approximately P (t1/2 = 180 min). It is suggested that, by analogy to the GTPase activity of p21(ras) and by examining the crystallographic structure of Spo0F, that the carboxyamide of the mutant Asn 56 may favorably position a catalytic water near the protein acyl phosphate to promote Spo0F approximately P K56N hydrolysis. It is also deduced that Lys 56 in the wild-type protein is critical for the efficient interaction and phosphoryl transfer between Spo0F and it's cognate protein kinase, KinA. Comparison of the known response regulators shows that inefficient autophosphatases (t1/2 on the order of hours) typically contain an amino acid residue with a long side chain at the position equivalent to 56 in Spo0F, whereas efficient autophosphatases (t1/2 on the order of minutes) frequently contain a residue with a carboxyamide or carboxylate side chain at this position. It appears that, by altering residues adjacent to the active site, the autophosphatase activity of response regulator proteins has been attenuated to match the diverse biological roles played by these proteins.     

Analysis of the stabilization of hen lysozyme by helix macrodipole and charged side chain interaction

In the N-terminal region of the alpha-helix of the c-type lysozymes, two Asx residues exist at the 18th and 27th positions. Hen lysozyme has Asp18/Asn27 (18D/27N), and we prepared three mutant lysozymes, Asn18/Asn27 (18N/27N), Asn18/Asp27 (18N/27D), and Asp18/Asp27 (18D/27D). The stability of the wild-type (18D/27N) lysozyme supported the existence of a hydrogen bond between the side chain of Asp18 and the amide group at the N1 position in the alpha-helix, while the stability of the 18N/27D lysozyme supported the presence of the capping box between the Ser24 (N-cap) and Asp27 residues. Although electrostatic repulsion was observed between Asp18 and Asp27 residues in 18D/27D lysozyme, the dissociation of each residue contributed to stabilizing the B-helix in 18D/27D lysozyme through hydrogen bonding and charge-helix macrodipole interaction. This is the first evidence that two neighboring negative charges at the N-terminus of the helix both increased the stability of the protein.     

Role of the active-site carboxylate in dihydrofolate reductase: kinetic and spectroscopic studies of the aspartate 26-->asparagine mutant of the Lactobacillus casei enzyme

A mutant of Lactobacillus casei dihydrofolate reductase, D26N, in which the active site aspartic acid residue has been replaced by asparagine by oligonucleotide-directed mutagenesis has been studied by NMR and optical spectroscopy and its kinetic behavior characterized in detail. On the basis of comparisons of a large number of chemical shifts and NOEs, it is clear that there are only very slight structural differences between the methotrexate complexes of the wild-type and mutant enzymes and that these are restricted to the immediate environment of the substitution. The data suggest a slight difference in orientation of the pteridine ring in the binding site in the mutant enzyme. Both NMR and UV spectroscopy show that methotrexate is protonated on N1 when bound to the wild-type enzyme but not when bound to the mutant. Binding constant measurements by fluorescence quenching and steady-state kinetic measurements of dihydrofolate (FH2) and folate reduction show that the substitution has little or no effect on substrate, coenzyme, and inhibitor binding (< 7-fold increase in Kd) and only a modest effect on kcat (up to a factor of 9 for FH2 and 25 for folate) and kcat/KM (up to a factor of 13 for FH2 and 14 for folate). Measurements of deuterium isotope effects and direct measurements of hydride ion transfer and product release by stopped-flow methods revealed that for the mutant enzyme hydride ion transfer is rate-limiting across the pH range 5-8. This allowed a direct comparison of the rate of hydride ion transfer in the wild-type and mutant enzymes; the asparagine substitution was found to decrease this rate by 62-fold at pH 5.5 and 9-fold at pH 7.5. This effect is much smaller than that seen for the corresponding mutant of Escherichia coli dihydrofolate reductase [Howell, E. E., Villafranca, J. E., Warren, M. S., Oatley, S. J., & Kraut, J. (1986) Science 231, 1123-1128], estimated as a 1000-fold decrease in the rate of hydride ion transfer. The change in pH dependence of kcat resulting from the substitution is consistent with, but does not prove, the idea that the group of pK 6.0 which must be protonated for hydride ion transfer to occur is Asp26. For folate reduction, the pH dependence of kcat is determined by two pKs, one of which, pK 5, disappears in the mutant enzyme, suggesting that it may correspond to ionization of Asp26.(ABSTRACT TRUNCATED AT 400 WORDS)     

Step-scan time-resolved FTIR spectroscopy of cytochrome P-450cam carbon monoxide complex: a salt link involved in the ligand-rebinding process

Step-scan time-resolved Fourier transform infrared spectroscopy with a time resolution of 5 micros was applied to the carbon monoxide complex of cytochrome P-450cam (CYP101) to study the bimolecular ligand-rebinding process after flash photolysis. Spectral changes in the CO ligand stretch vibration band and in the protein amide I' band were monitored simultaneously. In substrate complexes having the camphor C-8, C-9, and C-10 methyl groups, rebinding of the ligand and the relaxation of the protein proceed at the same rate within experimental errors. For substrate complexes missing the methyl groups, the relaxation fo the protein tends to relax slightly faster than the CO ligand rebinding to the heme iron. compared to the (1R)-camphor and the camphane complex, the bimolecular rebinding rate constant for P-450 bound with substrates lacking the methyl groups are increased by a factor of 10-40. An unusual signal at about 1719 cm-1 was found in the difference spectrum of the photolyzed minus nonphotolyzed CO complex which has not ben reported for other heme proteins so far. This signal is strongly pronounced in wild-type P-450cam bound with (1R)-camphor or camphane and in the D251N mutant bound with (1R)-camphor. In contrast, substrate-free P-450 and the norbornane and norcamphor complexes reveal only a very weak signal or a changed band shape. On the basis of the crystal structure data, we suggest that this signal originates from the rearrangement of the hydrogen-bonding pattern or the protonation state of the salt link between Asp297, Arg299, and the heme propionate group.     

Dynamics of protein-bound water in the heme domain of P450BM3 studied by high-pressure spectroscopy: comparison with P450cam and P450 2B4

Pressure-induced transitions in the heme domain of cytochrome P450BM3 (P450BMP) were studied versus the concentration of palmitic acid. An increase in hydrostatic pressure causes a high- to low-spin shift and subsequent P450 to P420 transition. Conversion of P450BMP to P420 is associated with important conformational and hydration changes of the protein. Treating the pressure-induced changes in the high-spin content in P450 in terms of the four-state model of spin transitions and substrate binding, we evaluated and compared the barotropic parameters of these transitions for P450MBP, P450cam, and P450 2B4 (2B4). In the current study, the pressure-induced transitions in P450cam were reinvestigated versus the concentration of camphor. The interactions of 2B4 and P450BMP with their substrates (benzphetamine and palmitic acid) were accompanied by larger changes in the partial volume of the proteins (+267 and +248 mL/mol, respectively) than the interactions of P450cam with camphor (+106 mL/mol). For 2B4 and P450BMP, substrate binding apparently requires hydration of regions outside the active site. The reaction volumes of the low- to high-spin transitions of the substrate-free cytochromes (20-23 mL/mol) are consistent with the displacement of one water molecule. The volume changes in the high- to low-spin transition of the substrate-bound P450cam, 2B4, and P450BMP (-90, -49, and -16 mL/mol correspondingly) reveal a linear relationship with DeltaG degrees of the spin transition, suggesting that modulation of the spin state by substrate binding is driven by a common mechanism in all three heme proteins.     

Probing the mechanism of the carcinogenic activation of N-nitrosodiethanolamine with deuterium isotope effects: in vivo induction of DNA single-strand breaks and related in vitro assays

A series of bioassays, including in vivo induction of DNA single-strand breaks (SSB) and cytotoxicity in cytochrome P450 2E1-transfected cells, were utilized with N-nitrosodiethanolamine (NDELA), its deuterated isotopomers (alpha-D4NDELA and beta-D4NDELA), N-nitroso-2-hydroxymorpholine (NHMOR), and two of its deuterated isotopomers (2-D-NHMOR and 5,5-D2-NHMOR) to probe the mechanism of carcinogenic activation of NDELA and the role of its metabolite NHMOR. DNA samples, taken from the livers of male Wistar rats 4 h after the administration of NDELA, exhibited dose-dependent DNA SSB levels over the range of 0.08-0.75 mmol/kg (body weight), with the greatest SSB level at the highest dose. Deuterium isotope effects on DNA SSB levels were inversely dependent on dose: alpha-D4NDELA, 3. 22-1.37; and beta-D4NDELA, 1.38-0.79. At the lowest dose of 0.15 mmol/kg (body weight), 5,5-D2-NHMOR gave an isotope effect for DNA SSB of 2.8 while that for 2-D-NHMOR was 0.7. NDELA and beta-D4NDELA were equally cytotoxic to human P450 2E1-transfected V79 Chinese hamster cells, while alpha-D4NDELA was not. Significant DNA SSB levels were observed in these cells for NDELA and beta-D4NDELA but not for alpha-D4NDELA. A kinetic deuterium isotope effect of 2.6 for Vmax/Km was observed for the horse liver alcohol dehydrogenase-mediated oxidation of beta-D4NDELA to NHMOR, while kH/kD for alpha-D4NDELA was 1.05. These data provide the first definitive evidence for the activation of NDELA by a pathway involving the scission of the alpha-CH bond and are consistent with P450 2E1-mediated alpha-hydroxylation of NDELA producing the corresponding reactive alpha-hydroxynitrosamine.     

Characterization of cytochrome P450TYR, a multifunctional haem-thiolate N-hydroxylase involved in the biosynthesis of the cyanogenic glucoside dhurrin

The haem-thiolate N-hydroxylase cytochrome P450TYR involved in the biosynthesis of the tyrosine-derived cyanogenic glucoside dhurrin in Sorghum bicolor had recently been isolated. Reconstitution of enzyme activity by insertion of cytochrome P450TYR and NADPH-cytochrome P450-reductase into L-alpha-dilauroylphosphatidylcholine micelles and using tyrosine as substrate results in the formation of p-hydroxyphenylacetaldehyde oxime. Quantitative substrate binding spectra demonstrate that tyrosine and N-hydroxytyrosine are mutually exclusive substrates that bind to the same active site of cytochrome P450TYR. The multifunctionality of cytochrome P450TYR has been confirmed in reconstitution experiments using recombinant cytochrome P450TYR expressed in Escherichia coli. It was earlier reported that an in vitro microsomal system catalyzing all but the last step in the biosynthetic pathway for cyanogenic glucosides exhibits catalytic facilitation (channelling). This observation is explained by the multifunctionality of cytochrome P450TYR. The cytochrome P450TYR sequence represents the first amino acid sequence of a functionally characterized cytochrome P-450 enzyme from a monocotyledonous plant and the first sequence of an N-hydroxylase with high substrate specificity. Multifunctional N-hydroxylases of the cytochrome P-450 type have not previously been reported in living organisms.