Leu Mutant Research Articles

1H NMR investigation of distal mutant deoxy myoglobins. Interpretation of proximal His contact shifts in terms of a localized distal water molecule.

1H NMR spectra of a series of distal point mutants of human and sperm whale deoxy myoglobin have been recorded and their spectral parameters compared with those of wild type. The substitutions investigated include His64(E7)-->Gly, Ala, Val, Leu, Ile, and Gln and Val68(E11)-->Ala, Ile. The three resonances from the proximal His F8 imidazole ring, as well as two heme methyl signals, are identified in each of the proteins. Significant perturbations of the NMR spectra of mutant deoxy myoglobins (Mbs) occurred only upon substitution of His64(E7) by any non-polar residue, with only minor variation in parameters throughout the range of side chains. These spectral changes are attributed to the elimination of a non-coordinated ordered water molecule in the distal pocket found hydrogen bonded to His64(E7) in crystals of wild-type deoxy Mb, but abolished in the His64(E7)-->Leu mutant deoxy Mb crystal (Quillin, M. L., Arduini, R. M., Olson, J. S., and Philips, G. N., Jr. (1993) J. Mol. Biol. 234, 140-155). The observed spectral changes, increased His F8 ring spin delocalization, and decreased heme in-plane asymmetry, can be directly attributed to the weakening of the effective axial field and a decrease in the asymmetry in the rhombic ligand field resulting from removal of the water molecule. The hyperfine shift patterns for the mutants His64(E7)-->Gln and Val68(E11)-->Ile deoxy Mbs are minimally perturbed from that of wild type and are interpreted to reflect a conserved distal water-binding site. In the point mutant Val68(E11)-->Ala, the decreased covalency to the axial His F8 is interpreted as reflecting a conserved distal water molecule that can interact more strongly with the iron due to the reduced steric bulk of the E11 side chain. The differential 1H NMR spectral parameters for the His F8 resonances in the two subunits of T state deoxy Hb A are shown to be similarly consistent with the known occupation of the distal water binding site in the alpha-, but not beta-subunit.

Mutants of Smooth Muscle Myosin Light Chain Kinase at Tryptophan 800

The importance of Trp 800 in the calmodulin-binding site of myosin light chain kinase was investigated. Truncation mutants from Leu 447 to the C-terminus were expressed in E. coli and these were modified by point mutations of Trp 800 to Gly, Cys, Leu and Tyr. Trp at this position was more effective than any of the other residues. The Leu mutant was partially active and its Km for calmodulin decreased from about 10 nM to 175 nM. The Tyr mutant had detectable activity but the other two mutants were inactive and did not bind calmodulin. Thus Trp at position 800 is critical. The activity of the Leu mutant at high calmodulin concentrations was less than the wild-type mutant, about 20%. This suggests that the binding of calmodulin does not release inhibition in an all-or-none mechanism and that other intramolecular interactions are important.

The effect of Met–>Leu mutations on calmodulin's ability to activate cyclic nucleotide phosphodiesterase.

Calmodulin (CaM) has two hydrophobic surface patches that are particularly rich in Met residues, and these are the major contact areas where CaM interacts with its target enzymes. The amino acid Leu has been introduced by site-directed mutagenesis to replace all the Met residues in CaM. All nine individual Met-->Leu mutants of CaM as well as some double and quadruple mutants were expressed in Escherichia coli. All mutants could be purified by calcium-dependent hydrophobic affinity chromatography, indicating that they still expose their hydrophobic surfaces upon binding calcium. Each single Met-->Leu mutation in the C-terminal domain of the protein had little effect on its ability to activate phosphodiesterase (PDE), while a quadruple mutant with four C-terminal Leu residues instead of Met has a significantly lower affinity for PDE. The M36L mutant is a poor activator compared with the other three N-terminal single Met-->Leu mutants, which have a slightly lower affinity for PDE than wild-type CaM. The introduction of a positively charged Arg for Met-145 resulted in an almost complete loss of CaM's ability to activate PDE. Nuclear magnetic resonance spectroscopy was used to show that most CaM mutants retain their overall three-dimensional structure. Thus, the altered activation properties appear to arise from differences in the flexibility and polarizability of the Met and Leu sidechains, rather than from structural perturbations.

Two-dimensional NMR Studies of Selenomethionyl Calmodulin

Calmodulin (CaM) is a ubiquitous calcium regulatory protein that can interact with almost 30 different target proteins. The majority of the CaM-binding domains of the target proteins are believed to interact with two hydrophobic surfaces on Ca2+-CaM; these two regions are very rich in Met residues. To obtain more information about the role of these residues, we have biosynthetically incorporated selenomethionine (SeMet) in place of the nine Met residues of CaM. Amino acid analysis shows that the SeMet-CaM contains 15% Met and 85% SeMet. SeMet-CaM retains many of the properties of the wild-type protein; it activates the enzyme cyclic nucleotide phosphodiesterase, it binds to phenyl-Sepharose and myosin light chain kinase (MLCK) in a calcium-dependent manner, and it experience a calcium-dependent band shift during SDS-gel electrophoresis. Moreover, by comparing the natural abundance (1H, 13C)-heteronuclear multiple quantum coherence (HMQC) spectra of the calcium, apo and target peptide-bound forms of wild-type CaM and SeMet-CaM, we have found that the two proteins have very similar, if not identical, structures. Thus, the substitution of SeMet for Met does not cause a change in the conformation and function of CaM, in agreement with the results obtained for other proteins. The apo, calcium and target peptide-bound forms of SeMet-CaM were subsequently studied by natural abundance (1H, 77 Se)-heteronuclear multiple bond correlation (HMBC) and (1H, 13C)-HMQC NMR. Nine well-resolved 77Se resonances could be observed. Substitution of SeMet for Met gave rise to the same 1H and 13C chemical shift changes for each individual Met residue, this facilitated making the assignments from known 1 H, 13C assignments of the Met residues. Some of these assignments were confirmed by studying Met→Leu mutants of CaM. With the exception of Met76, which always remains solvent exposed, all resonances experienced large 77Se chemical shift changes upon the addition of Ca2+ and the MLCK peptide. The large shift changes indicate that the electron distribution in the SeMet side-chain can be adjusted for the different states of CaM, suggesting that the polarizability of sulfur or selenium may be important for the proper functioning of CaM. This study also shows that the natural abundance (1H, 77Se)-HMBC experiment provides a sensitive approach for the study of SeMet protein

Crystal Structures of Escherichia coli Aspartate Aminotransferase in Two Conformations: Comparison of an Unliganded Open and Two Liganded Closed Forms

Three crystal structures of wild type E. coli aspartate aminotransferase (E.C.2.6.1.1) in space group P21 have been determined at resolution limits between 2·6 and 2·35 Å. The unliganded enzyme and its complexes with the substrate analogues maleate and 2-methylaspartate resulted in different conformations. The unit cell parameters of the unliganded and the inhibited enzyme are a = 87·2, b = 79·9, c = 89·8 Å and β = 119·1°, and a = 85·4, b = 79·8, c = 89·5 Å and β = 118·6°, respectively. The crystallographic symmetry is pseudo-C2221. The liganded enzyme structures were solved by difference Fourier techniques from that of VAL39→ Leu mutant partially refined an R-factor of 0·22 at 2·85 Å. They have a "closed" conformation like the chicken mAATase: maleate complex. The models were refined to R-factors of 0·19 (maleate complex) and 0·18 (2-methylaspartate complex) by molecular dynamics and restrained least squares methods. The unliganded crystal form was solved by molecular replacement and refined to an R-factor of 0·19 at 2·5 Å resolution. The structure is in a "half-open" conformation, with the small domain rotated about 6° from the closed conformation. The cofactor pyridoxal phosphate has a more relaxed conformation than in mAATase. Both maleate and 2-methylaspartate are hydrogen-bonded to the active site as in mAATase. The C-α-CH3 bond of 2-methylaspartate is oriented at right angles to the cofactor pyridine ring, the most productive orientation for α-deprotonation of the substrate l-aspartate. Comparisons with earlier determined eAATase structures in space group C 2221 revealed differences that can probably be attributed to the somewhat lower resolution of the orthorhombic structures and/or mutations in the eAATases used in those studies. The present P21 structures confirm the justification of extrapolating properties of active site point mutants to the vertebrate isozymes. They will serve as reference in the interpretation of the properties of further site-directed mutants in continued studies of structure-function relationships of this enzyme.

Molecular Engineering of Horseradish Peroxidase. Highly Enantioselective Sulfoxidation of Aryl Alkyl Sulfides by the Phe-41 .fwdarw. Leu Mutant

Molecular Engineering of Horseradish Peroxidase. Highly Enantioselective Sulfoxidation of Aryl Alkyl Sulfides by the Phe-41 .fwdarw. Leu Mutant

The hydration of Ras p21 in solution during GTP hydrolysis based on solution X-ray scattering profile.

The small-angle X-ray scattering technique was used to characterize the structure in solution of wild type ras p21 as well as the oncogenic proteins mutated at residue 12, 59, or 61. In the presence of GDP, the radius of gyration, Rg, determined for wild type ras p21 was 16.89 +/- 0.01 A, while the wild type ras p21 bound to the GTP analogue GDPNHP (5'-guanyl imido diphosphate beta-gamma-imidoguanosine 5'-triphosphate) showed an Rg value of 17.46 +/- 0.01 A, which is 3.3% larger. The result shows that ras p21 expands upon GTP binding. The Rgs of mutated proteins were 17.04 +/- 0.01, 16.98 +/- 0.01, and 17.03 +/- 0.01 A for the Gly-12 to Val, Ala-59 to Thr, and Gln-61 to Leu mutants, respectively. The scattering profiles were analyzed by simulation of hydrated ras p21, based on the crystal atomic coordinates, and it was concluded that the ras p21 molecule incorporates 20% more bulk water upon GTP binding. The increase of bulk water is especially conspicuous around the interface between switch I (residues 32-40) and switch II (residues 60-66) regions. This suggests that hydration plays an important role in the interaction with GAP.

Structural determinants of cytochrome P450 2B1 specificity: evidence for five substrate recognition sites.

Twelve site-directed mutants of rat cytochrome P450 2B1 distributed over seven positions and four putative substrate recognition sites (SRS) were constructed and expressed in COS cells. Function was examined using androstenedione and testosterone as substrates. Substitutions at positions 303, 360, and 473 did not markedly affect the regio- or stereoselectivity of androgen metabolism, whereas mutants in positions 206 (SRS-2), 302 (SRS-4), and 363 and 367 (SRS-5) exhibited markedly different steroid metabolite profiles compared with parental P450 2B1. In particular, the Phe-206-->Leu substitution conferred androgen 6 alpha- and testosterone 7 alpha-hydroxylase activities, and the Thr-302-->Ser substitution suppressed androgen 16 beta-hydroxylation in favor of androstenedione 16 alpha- and testosterone 15 alpha-hydroxylation. Replacement of Val-363 or Val-367 with Ala conferred androgen 15 alpha-hydroxylase and 6 beta-hydroxylase activities, respectively, and suppressed susceptibility to mechanism-based inactivation by the P450 2B1-selective chloramphenicol analog N-(2-p-nitrophenethyl)chlorofluoroacetamide. The Val-367-->Ala mutant was also resistant to chloramphenicol itself. The Leu mutant at position 363 exhibited increased specificity for androstenedione and testosterone 16 beta-hydroxylation, whereas the Leu mutant at position 367 exhibited decreased stereospecificity. Most interestingly, the size of key residues identified plays a critical role in governing steroid hydroxylation from the alpha-face or beta-face and hydroxylation on the D-ring or the B-ring.(ABSTRACT TRUNCATED AT 250 WORDS)

Putative functions of phenylalanine-350 of Pseudomonas putida cytochrome P-450 cam

Cytochrome P-450 cam hydroxylates d-camphor, using molecular oxygen and reducing equivalents transferred via putidaredoxin. We constructed mutant genes in which Phe-350 of P-450 cam was replaced by Leu, Tyr, or His by site-directed mutagenesis, expressed them in Escherichia coli, purified the mutant proteins, and compared their enzymic properties with those of the wild type P-450 cam. NADH oxidation rate of the Tyr mutant in the reconsituted system with putidaredoxin and putidaredoxin reductase was similar to that of the wild type enzyme, while the Leu mutant and the His mutant showed 67% and 17% activity of that of the wild type, respectively. The affinities of these mutant proteins for camphor and the oxidized form of putidaredoxin were much the same as those of the wild type protein. Rate constants for the reduction reaction of P-450 cam by reduced putidaredoxin, a physiological electron donor for P-450 cam, of Tyr and His mutants were much the same as that of the wild type enzyme, whereas the Leu mutant showed approx. half that of the wild type. Thus, the aromatic ring of Phe-350 of P-450 cam probably contributes to enhancing efficiency of the electron transfer yet does not seem to be essential for the reaction.

Mutation of histidine 373 to leucine in cytochrome P450c17 causes 17 alpha-hydroxylase deficiency.

We identified a new homozygous missense mutation His373-->Leu in the CYP17 gene of two sisters with 17 alpha-hydroxylase deficiency with an elevated plasma aldosterone concentration by sequencing their genomic DNAs amplified by polymerase chain reaction. Using polymerase chain reaction-based site-directed mutagenesis, we prepared a DNA that encoded the Leu373 mutant protein. COS-1 cells transfected with the mutant DNA, despite having an RNA hybridizable to the P450c17 cDNA, did not show 17 alpha-hydroxylase and 17,20-lyase activities. Also, the cells were devoid of 11 beta-hydroxylase and aldosterone synthase activities. To examine the mechanism by which the single amino acid change His373-->Leu eliminates activity, we expressed N-terminally modified P450c17 proteins with and without the Leu373 mutation in Escherichia coli and performed spectral studies. Membrane preparations from E. coli cells expressing the wild-type form of the modified enzyme showed an absorption peak at 449 nm upon addition of carbon monoxide in the reduced state and produced characteristic substrate-induced difference spectra, whereas those from the cells expressing the mutant form did not show these spectral changes. The 17 alpha-hydroxylase and 17,20-lyase activities were observed only in E. coli cells expressing the wild-type enzyme. These results show that the His373-->Leu mutant does not incorporate the heme prosthetic group properly and suggest a critical role of His373 in heme binding.

Triplet state EPR of reaction centers from the HisL173?LeuL173 mutant of Rhodobacter sphaeroides which contains a heterodimer primary donor

Electron paramagnetic resonance (EPR) spectroscopy has been used to examine the triplet states in reaction centers of Rhodobacter sphaeroides which have undergone a genetic modification affecting the primary donor. Reaction centers containing the His(L173)→Leu(L173) substitution in the amino acid sequence have a primary donor which consists of a BChl-BPh heterodimer. The triplets formed in this heterodimer reaction center were compared with those formed in the wild-type reaction center which contains the BChl-BChl homodimer. Both reaction centers transfer triplet energy to the carotenoid under illumination at liquid nitrogen temperatures (∼90 K). However, the intensity of the carotenoid triplet signal is significantly decreased in the Leu(L173) mutant compared with the wild-type reaction center. At 12 K, in wild-type reaction centers only the primary donor triplet is observed. The Leu(L173) mutant exhibits a signal similar to that observed by Bylina et al. (1990) in His(M200)→Leu(M200) mutant reaction centers from Rb. capsulatus. The values of the zero-field splitting parameters of this triplet are discussed within the context of various models for the primary donor triplet state. No alteration in the ability of the carotenoid to quench the primary donor triplet state results from mutations at these sites.

Mutations designed to modify the environment of the primary electron donor of the reaction center from Rhodobacter sphaeroides: phenylalanine to leucine at L167 and histidine to phenylalanine at L168.

Two mutations, L168 His to Phe and L167 Phe to Leu, were made in residues near the primary electron donor, a bacteriochlorophyll dimer, of the reaction center from Rhodobacter sphaeroides. Blue shifts of 10-15 nm in the 865-nm band of the donor were observed in the optical absorption spectra of both of the mutant reaction centers. The rate of initial electron transfer was determined by measurement of the kinetics of the decay of the excited state of the donor, and the rate of charge recombination was determined by measurement of the recovery of the bleaching of the donor. The initial electron transfer time constant and the charge recombination time constant were determined to be 3.6 ps and 220 ms, respectively, in the L168 His to Phe mutant and 5.0 ps and 85 ms in the L167 Phe to Leu mutant, compared to 3.8 ps and 100 ms measured for the wild type. The oxidation potential of the donor measured by oxidation-reduction titrations was found to decrease by 80 mV in the L168 His to Phe mutant and increase by 25 mV in the L167 Phe to Leu mutant. Time-resolved fluorescence decay measurements indicated that the change in the oxidation potential of the donor in the L168 His to Phe mutant resulted in a change in the energies of the charge-separated states. The results show that an increase in the driving force does not increase the rate of the initial electron transfer reaction.(ABSTRACT TRUNCATED AT 250 WORDS)

The short loop between epidermal growth factor-like domains 4 and 5 is critical for human thrombomodulin function.

Thrombomodulin (TM) is a cofactor for activation of protein C by thrombin. We showed that 80-90% of this cofactor activity is lost by oxidation of Met388, located within the short interdomain loop between epidermal growth factor-like domains 4 and 5 (Glaser, C. B., Morser, J., Clarke, J. H., Blasko, E., McLean, K., Kuhn, I., Chang, R.-J., Lin, J.-H., Vilander, L., Andrews, W. H., and Light, D. R. (1992) J. Clin. Invest. 90, 2565-2573). For each of the 3 amino acids of the loop, site-specific mutants are described in which, 1) all possible single amino acid substitutions are made, 2) deletions are made, or 3) alanine is inserted adjacent to each residue of the loop. Most substitutions within the loop (38/57) result in a > 50% decrease in cofactor activity, while changes in the length of this region result in > 90% loss of activity. Only the Met388-->Leu mutant has higher cofactor activity (2-fold) than wild-type TM. A number of soluble and full-length TM analogs with the Met388-->Leu substitution are improved thrombin cofactors, whether produced in bacteria, insect, or mammalian cells. Detailed kinetic analysis of a soluble TM analog consisting of the six EGF-like domains secreted from insect cells shows that the enhanced activity of the Met388-->Leu mutant results from an increased catalytic efficiency (kcat/Km). This enhancement is maximal at physiological concentrations of calcium. The loss of activity following Met388 oxidation in the wild-type protein is the result of both decreased binding to thrombin (Kd effect) and a decreased interaction of the TM.thrombin complex with protein C (Km effect). We demonstrate the critical role of this interdomain loop in the biological anticoagulant properties of TM.

Site-directed mutagenesis of GLUT1 in helix 7 residue 282 results in perturbation of exofacial ligand binding.

The structure-function relationship of the HepG2/erythrocyte-type glucose transporter (GLUT1) has been studied by in vitro site-directed mutagenesis. Chinese hamster ovary clones in which glucose transporters were transfected were shown by Western blotting with a GLUT1 anti-COOH-terminal peptide antibody to have expression levels of Gln282----Leu, Asn288----Ile, and Asn317----Ile mutations that were comparable with the wild type. All three mutant GLUT1 clones had high 2-deoxy-D-glucose transport activity compared with a nontransfected clone, suggesting that these residues are not absolutely required for the transport function. We have examined the possibility that the inner and outer portions of the transport pathway are structurally separate by measuring the interaction of the mutant transporters with the inside site-specific ligand cytochalasin B and the outside site-specific ligand 2-N-4-(1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis(D-mannos-4 -yloxy)-2- propylamine (ATB-BMPA). All three mutant GLUT1 clones showed high levels of cytochalasin B labeling, and the N288I and N317I mutants showed high levels of ATB-BMPA labeling. In contrast to the transport and cytochalasin B labeling results, the transmembrane helix 7 Gln282----Leu mutant was labeled by ATB-BMPA to a level that was only 5% of the level observed in the wild type. We have confirmed that this mutant was defective in the outer site by comparing the inhibition of wild-type and mutant 2-deoxy-D-glucose transport by the outside site-specific ligand 4,6-O-ethylidene-D-glucose. 4,6-O-Ethylidene-D-glucose inhibited wild-type transport with a Ki of approximately 12 mM, but this was increased to greater than 120 mM in the Gln282----Leu mutant. Thus, of the 3 residues mutated in this study, only glutamine 282 substitution causes a major perturbation in function, and this is a specific and striking reduction in the affinity for the outside site-specific ligands ATB-BMPA and 4,6-O-ethylidene-D-glucose.

Role of residue 478 as a determinant of the substrate specificity of cytochrome P450 2B1.

Two allelic variants and eight site-directed mutants of cytochrome P450 2B1 differing at residue 478 have been expressed in COS cells and assayed for androstenedione hydroxylase activities. The 478Gly and 478Ala variants and five mutants (Ser, Thr, Val, Ile, and Leu) exhibited 16 beta-OH:16 alpha-OH ratios ranging from 0.7 to 9.3, whereas the Pro, Glu, and Arg mutants were expressed but inactive. The seven samples active toward androstenedione also exhibited testosterone 16 beta-OH:16 alpha-OH ratios ranging from 0.4 to 2.3. With both steroids, the Gly variant had the highest 16 beta-hydroxylase activity, and the 16 beta-OH:16 alpha-OH ratio increased with the size of aliphatic size chains (Ala, Val, and Ile/Leu). The highest ratio of androgen 15 alpha:16-hydroxylation was observed with the Ser mutant. On the basis of previous work indicating decreased susceptibility of the 478Ala variant in liver microsomal and reconstituted systems to inactivation by chloramphenicol analogs, methodology was refined for monitoring enzyme inactivation in COS cell microsomes. The Gly and Ala variants were inactivated by chloramphenicol with similar rate constants, whereas the Ser and Val mutants were inactivated more slowly, and the Leu mutant was refractory. Only the Gly variant was inactivated by the chloramphenicol analog N-(2-p-nitrophenethyl)chlorofluoroacetamide. Thus, the side chain of residue 478 appears to be a major determinant of enzyme inactivation as well as of androgen hydroxylation.(ABSTRACT TRUNCATED AT 250 WORDS)

Investigating interdomain region mutants Phe194----Leu and Phe194----Trp of yeast phosphoglycerate kinase by 1H-NMR spectroscopy.

Site-directed mutagenesis has been used to produce two mutant forms of yeast phosphoglycerate kinase in which the interdomain residue Phe194 has been replaced by a leucine or tryptophan residue. Using 1H-NMR spectroscopy, it was found that the mutations at position 194 induce both local and long-range conformational changes in the protein. It was also found that 3-phosphoglycerate binding to the mutant proteins induces somewhat different conformational effects to those observed for wild-type phosphoglycerate kinase. The affinity of mutant Phe194----Trp for 3-phosphoglycerate was found by NMR studies to be unaffected, while the affinity of Phe194----Leu mutant is reduced by about threefold relative to the wild-type enzyme. The binding of ATP at the electrostatic site of the mutant proteins is also seen to be about three times weaker for the Phe194----Leu mutant when compared to wild-type or Phe194----Leu mutant. These results are discussed in the light of the kinetic studies on the mutants which show that for Phe194----Leu mutant the Km values for both 3-phosphoglycerate and ATP, as well as the Vmax, are decreased relative to the wild-type enzyme, while for mutant Phe194----Trp, the Km values for 3-phosphoglycerate and ATP are unaffected and the Vmax is decreased when compared to wild-type enzyme. Kinetic studies in the presence of sulphate reveal that the anion activation is greater for mutant Phe194----Trp and less for mutant Phe194----Leu, relative to that observed for wild-type phosphoglycerate kinase. The NMR data, taken together with the kinetic data, are consistent with the on and off rates of 3-phosphoglycerate being affected by the mutations at position 194. It is suggested that Phe194 is important for the mobility of the interdomain region and the relative movement of the 3-phosphoglycerate binding site which allows the optimum conformation for catalysis to be attained. Apparently Trp194 reduces the mobility of the interdomain region of the protein, while Leu194 increases it.

EPR characterization of the stereochemistry of the distal heme pocket of the engineered human myoglobin mutants.

Recombinant human myoglobin mutants with the distal histidine residue replaced by Leu, Val, or Gln residues have been prepared by site-directed mutagenesis and expression in Escherichia coli. The recombinant apomyoglobin proteins have been successfully reconstituted with cobaltous protoporphyrin IX to obtain cobalt myoglobin mutant proteins, and the role of the distal histidine residue on the interaction between the bound ligand and the myoglobin molecule has been studied by EPR spectroscopy. We found that the distal histidine residue is significant in the orientation of the bound oxygen molecule. Low temperature photolysis experiments on both oxy cobalt proteins and ferric nitric oxide complexes indicated that the nature of the photolyzed form depends on the steric crowding of the distal heme pocket. To our surprise, the distal Leu mutant has a less restricted, less sterically crowded distal heme pocket than that of the distal Val mutant myoglobin, despite the fact that Leu has a larger side chain volume than Val. Our results demonstrate that the distal heme pocket steric crowding is not necessarily related to the side chain volume of the E7 residue.

Conformational change and histidine control of heme chemistry in cytochrome c peroxidase: resonance Raman evidence from Leu-52 and Gly-181 mutants of cytochrome c peroxidase.

Resonance Raman (RR) spectra are reported for Fe(III), Fe(II), and Fe(II)CO forms of site-directed mutants of the cytochrome c peroxidase variant CCP(MI), cloned in Escherichia coli. The Fe(II) form is five-coordinate (5-c) and high-spin at low pH, but it is six-coordinate (6-c) and low-spin at high pH except when the distal His-52 residue is replaced with Leu, showing the sixth ligand to be the His-52 imidazole. Although the Leu-52 mutant stays 5-c, it does undergo an alkaline transition, as revealed by upshifts and broadening of bands assigned to vinyl C = C stretching (1620 cm-1) and C beta-vinyl bending (402 cm-1). Similar changes are seen for CCP(MI) and other mutants. Thus the alkaline transition induces a conformational change that affects the vinyl groups, probably through changes in their orientation, and that permits the His-52 imidazole to bind the Fe. The RR band arising from the stretching of the proximal Fe(II)-imidazole bond contains components at ca. 235 and 245 cm-1 for CCP(MI), which are believed to reflect a double well potential for the H-bond between the proximal His-175 imidazole and the Asp-235 carboxylate group. Loss of this H-bond by mutation of Asp-235 to Asn results in the loss of these two bands and their replacement by a single band at 205 cm-1. Although the Fe(II)-imidazole stretching mode cannot be observed in the 6-c alkaline form of the enzyme, the sixth ligand in the alkaline form of CCP(MI) is photolabile, and the status of the Fe(II)-imidazole bond can be determined in the resulting 5-c-photoproduct. For CCP(MI) at alkaline pH, the conformation change induces an increase in the 235/245-cm-1 ratio, reflecting a perturbation of the H-bond potential. In the His-52----Leu mutant, a 205-cm-1 band appears along with the 235/245-cm-1 doublet at alkaline pH, indicating partial loss of the proximal H-bond due to the distal alteration. The effect of mutations that perturb the H-bonding network that extends from the distal to the proximal side of the heme is more dramatic: at alkaline pH, His-181----Gly, Arg-48----Leu, and Trp-51----Phe mutants show an Fe(II)-imidazole stretching mode at 205 cm-1 exclusively, indicating complete loss of the proximal Asp-235-His-175 H-bond.(ABSTRACT TRUNCATED AT 400 WORDS)

Role of lysine 173 in heparin binding to heparin cofactor II

Heparin cofactor II (HC) is a plasma serine proteinase inhibitor (serpin) that inhibits alpha-thrombin in a reaction that is dramatically enhanced by heparin and other glycosaminoglycans/polyanions. We investigated the glycosaminoglycan binding site in HC by: (i) chemical modification with pyridoxal 5'-phosphate (PLP) in the absence and presence of heparin and dermatan sulfate; (ii) molecular modeling; and (iii) site-directed oligonucleotide mutagenesis. Four lysyl residues (173, 252, 343, and 348) were protected from modification by heparin and to a lesser extent by dermatan sulfate. Heparin-protected PLPHC retained both heparin cofactor and dermatan sulfate cofactor activity while dermatan sulfate-protected PLPHC retained some dermatan sulfate cofactor activity and little heparin cofactor activity. Molecular modeling studies revealed that Lys173 and Lys252 are within a region previously shown to contain residues involved in glycosaminoglycan binding. Lys343 and Lys348 are distant from this region, but protection by heparin and dermatan sulfate might result from a conformational change following glycosaminoglycan binding to the inhibitor. Site-directed mutagenesis of Lys173 and Lys343 was performed to further dissect the role of these two regions during HC-heparin and HC-dermatan sulfate interactions. The Lys343----Asn or Thr mutants had normal or only slightly reduced heparin or dermatan sulfate cofactor activity and eluted from heparin-Sepharose at the same ionic strength as native recombinant HC. However, the Lys173----Gln or Leu mutants had greatly reduced heparin cofactor activity and eluted from heparin-Sepharose at a significantly lower ionic strength than native recombinant HC but retained normal dermatan sulfate cofactor activity. Our results demonstrate that Lys173 is involved in the interaction of HC with heparin but not with dermatan sulfate, whereas Lys343 is not critical for HC binding to either glycosaminoglycan. These data provide further evidence for the determinants required for glycosaminoglycan binding to HC.

Papain-Inhibitory Activity of Oryzacystatin, a Rice Seed Cysteine Proteinase Inhibitor,Depends on the Central Gln-Val-Val-Ala-Gly Region Conserved among Cystatin Superfamily Members

Oryzacystatin, a cysteine proteinase inhibitor occurring in rice seeds, contains a particular glycine residue (Gly5) near the NH2-terminal position, and the sequence Gln53-Val54-Val55-Ala56-Gly57 in a central part of the molecule. Both are conserved among most members of the cystatin superfamily. We have found from Escherichia coli expression studies that the NH2-terminal 21 residues of oryzacystatin are not essential for its papain-inhibitory activity, and that the conserved pentapeptide region may be indispensable [Abe, K., Emori, Y., Kondo, H., Arai, S., & Suzuki, K. (1988) J. Biol. Chem. 263, 7655-7659]. Here we present more detailed data based on quantitative analyses of the inhibitory activities of NH2- and COOH-terminally truncated oryzacystatin and site-directed mutants at the Gln-Val-Val-Ala-Gly region. The data indicate the following results. (1) The truncated mutants lacking the NH2-terminal 21 residues or the COOH-terminal 11 residues exhibit potent papain-inhibitory activity equivalent to the activity of wild oryzacystatin. (2) However, neither the mutant lacking the NH2-terminal 38 residues nor that lacking the COOH-terminal 35 residues is completely able to inhibit papain. (3) Site-directed mutants at the Gln residue of the Gln-Val-Val-Ala-Gly region have drastically reduced papain-inhibitory activities: the Gln----Pro mutant is completely inactive and the Gln----Leu mutant has an approximately 150 times higher Ki value than wild-type oryzacystatin.(ABSTRACT TRUNCATED AT 250 WORDS)