Structure and function of the catalytic site mutant Asp 99 Asn of phospholipase A2: absence of the conserved structural water.

To probe the role of the Asp-99 ... His-48 pair in phospholipase A2 (PLA2) catalysis, the X-ray structure and kinetic characterization of the mutant Asp-99-->Asn-99 (D99N) of bovine pancreatic PLA2 was undertaken. Crystals of D99N belong to the trigonal space group P3(1)21 and were isomorphous to the wild type (WT) (Noel JP et al., 1991, Biochemistry 30:11801-11811). The 1.9-A X-ray structure of the mutant showed that the carbonyl group of Asn-99 side chain is hydrogen bonded to His-48 in the same way as that of Asp-99 in the WT, thus retaining the tautomeric form of His-48 and the function of the enzyme. The NH2 group of Asn-99 points away from His-48. In contrast, in the D102N mutant of the protease enzyme trypsin, the NH2 group of Asn-102 is hydrogen bonded to His-57 resulting in the inactive tautomeric form and hence the loss of enzymatic activity. Although the geometry of the catalytic triad in the PLA2 mutant remains the same as in the WT, we were surprised that the conserved structural water, linking the catalytic site with the ammonium group of Ala-1 of the interfacial site, was ejected by the proximity of the NH2 group of Asn-99. The NH2 group now forms a direct hydrogen bond with the carbonyl group of Ala-1.     

Substitutions in the active site of chloramphenicol acetyltransferase: role of a conserved aspartate

The role of conserved Asp-199 in chloramphenicol acetyltransferase (CAT) has been investigated by site-directed mutagenesis. Substitution of Asp-199 by alanine results in a thermolabile mutant enzyme (Ala-199 CAT) with reduced kcat(13-fold) but similar Km values to wild type CAT. Replacement by asparagine gives rise to a thermostable mutant enzyme (Asn-199 CAT) with much reduced kcat(1500-fold). Furthermore, Asn-199 CAT shows anomalous inactivation kinetics with the affinity reagent 3-(bromo-acetyl)chloramphenicol. These results favor a structural role for Asp-199 rather than a catalytic one, in keeping with crystallographic evidence for involvement of Asp-199 in a tight salt bridge with Arg-18. Replacement of Arg-18 by valine results in a mutant enzyme (Val-18 CAT) with similar properties to Ala-199 CAT. The catalytic imidazole of His-19 appears to be conformationally constrained by hydrogen bonding between N1-H and the carbonyl oxygen of the same residue and by ring stacking with Tyr-25.     

Intramolecular interactions in chemically modified Escherichia coli thioredoxin monitored by hydrogen/deuterium exchange and electrospray ionization mass spectrometry.

Site specific amide hydrogen/deuterium content of oxidized and reduced Escherichia colithioredoxin, and alkylated derivatives, Cys-32-ethylglutathionylated and Cys-32-ethylcysteinylated thioredoxins are measured, after exposure for 20 s to D(2)O/phosphate buffer (pH 5.7), by electrospray mass spectrometry. The degree of deuteration of Oxi-TRX and Red-TRX correlated with the rates of H/D exchange measured previously by NMR. The ethylcysteinyl modification was shown to minimally perturb the active site of the reduced protein, but showed more global effects on structures of alpha-helices and beta-strands distant from the site of modification. In contrast, the larger ethylglutathionyl group had little effect on the protein's overall conformation, but significantly affected the structure of loops close to the active site. A molecular model of GS-ethyl-TRX derived from molecular simulation allowed the H/D exchange results to be interpreted in terms of specific interactions between the alkyl chain and the protein surface. The specific conformation of the ethylglutathione modification was predicted to be fixed by salt bridges between the carboxylates of the gamma-Glu and Gly of glutathione and the guanidinium of Arg-73 and epsilon-amino group of Lys-90 of the protein. Specific hydrogen bonding interactions between the glutathione carbonyl oxygens and the amide protons of thioredoxin residues Ile-75 and Ala-93 were predicted. The H/D exchange studies showed low levels of deuterium incorporation at backbone nitrogens of these residues. The data also provided evidence for an unusual amide proton-amide nitrogen hydrogen bond within the ethylglutathionylated chain. These same sets of electrostatic and hydrogen bonding interactions were not predicted or observed for the smaller alkyl modification in Cys-ethyl-TRX.     

Correlations between kinetic and X-ray analyses of engineered enzymes: crystal structures of mutants Cys----Gly-35 and Tyr----Phe-34 of tyrosyl-tRNA synthetase

The crystal structures of two mutant tyrosyl-tRNA synthetases (TyrTS) are reported to test predictions from kinetic data about structural perturbations and also to aid in the interpretation of apparent strengths of hydrogen bonds measured by protein engineering. The enzyme-tyrosine and enzyme-tyrosyl adenylate complexes of the mutant, TyrTS(Cys----Gly-35), have been determined at 2.5- and 2.7-A resolution, respectively. Residue Cys-35 is in the ribose binding site. Small rearrangements in structure are seen in the enzyme-tyrosine complex that are localized around the cavity created by the mutation. The side chain of Thr-51 moves to occupy the cavity, and Ile-52 adopts two significantly populated conformations, one as in the native enzyme and a second unique to the mutant. On binding tyrosyl adenylate, Ile-52 in the mutant crystal structure preferentially occupies the conformation observed in the native structure. The side chain at Thr-51 becomes disordered. The double-mutant test, which was designed to detect interactions between residues, had previously shown a discrepancy of some 0.4 kcal/mol on mutating Cys-35 and Thr-51 separately and together. A crystal structure of a second mutant, delta TyrTS(Tyr----Phe-34), complexed with tyrosine has been determined at 2.7-A resolution. Tyr-34 in wild-type enzyme makes a hydrogen bond with the phenolic oxygen of the bound tyrosine substrate. The mutant crystal structure was solved to discover whether or not a water molecule binds to the substrate instead of the hydroxyl of Tyr-34 as the interpretation of apparent binding energies from site-directed mutagenesis experiments hinges crucially on whether there is access of water to the mutated region.     

Effects of mutations of conserved Lys-155 and Thr-156 residues in the phosphate-binding glycine-rich sequence of the F1-ATPase beta subunit of Escherichia coli.

beta Lys-155 in the glycine-rich sequence of the beta subunit of Escherichia coli F1-ATPase has been shown to be near the gamma-phosphate moiety of ATP by affinity labeling (Ida, K., Noumi, T., Maeda, M., Fukui, T., and Futai, M. (1991) J. Biol. Chem. 266, 5424-5429). For examination of the roles of beta Lys-155 and beta Thr-156, mutants (beta Lys-155-->Ala, Ser, or Thr; beta Thr-156-->Ala, Cys, Asp, or Ser; beta Lys-155/beta Thr-156-->beta Thr-155/beta Lys-156; and beta Thr-156/beta Val-157-->beta Ala-156/beta Thr-157) were constructed, and their properties were studied extensively. The beta Ser-156 mutant was active in ATP synthesis and had approximately 1.5-fold higher membrane ATPase activity than the wild type. Other mutants were defective in ATP synthesis, had < 0.1% of the membrane ATPase activity of the wild type, and showed no ATP-dependent formation of an electrochemical proton gradient. The mutants had essentially the same amounts of F1 in their membranes as the wild type. Purified mutant enzymes (beta Ala-155, beta Ser-155, beta Ala-156, and beta Cys-156) showed low rates of multisite (< 0.02% of the wild type) and unisite (< 1.5% of the wild type) catalyses. The k1 values of the mutant enzymes for unisite catalysis were lower than that of the wild type: not detectable with the beta Ala-156 and beta Cys-156 enzymes and 10(2)-fold lower with the beta Ala-155 and beta Ser-155 enzymes. The beta Thr-156-->Ala or Cys enzyme showed an altered response to Mg2+, suggesting that beta Thr-156 may be closely related to Mg2+ binding. These results suggest that beta Lys-155 and beta Thr-156 are essential for catalysis and are possibly located in the catalytic site, although beta Thr-156 could be replaced by a serine residue.     

Unpaired cysteine-54 interferes with the ability of an engineered disulfide to stabilize T4 lysozyme

We have introduced an intramolecular disulfide bond into T4 lysozyme and have shown this molecule to be significantly more stable than the wild-type molecule to irreversible thermal inactivation [Perry, L.J., & Wetzel, R. (1984) Science (Washington, D.C.) 226, 555-557]. Wild-type T4 lysozyme contains two free cysteines, at positions 54 and 97, and no disulfide bonds. By directed mutagenesis of the cloned T4 lysozyme gene, we replaced Ile-3 with Cys. Oxidation in vitro generated an intramolecular disulfide bond; proteolytic mapping showed this bond to connect Cys-3 to Cys-97. While this molecule exhibited substantially more stability against thermal inactivation than wild type, its stability was further enhanced by additional modification with thiol-specific reagents. This and other evidence suggest that at basic pH and elevated temperatures Cys-54 is involved in intermolecular thiol/disulfide interchange with the engineered disulfide, leading to inactive oligomers. Mutagenic replacement of Cys-54 with Thr or Val in the disulfide-cross-linked variant generated lysozymes exhibiting greatly enhanced stability toward irreversible thermal inactivation.     

Effect of an engineered disulfide bond on the folding of T4 lysozyme at low temperatures

Equilibrium and kinetic effects on the folding of T4 lysozyme were investigated by fluorescence emission spectroscopy in cryosolvent. To study the role of disulfide cross-links in stability and folding, a comparison was made with a mutant containing an engineered disulfide bond between Cys-3 (Ile-3 in the wild type) and Cys-97, which links the C-terminal domain to the N terminus of the protein [Perry & Wetzel (1984) Science 226, 555]. In our experimental system, stability toward thermal and denaturant unfolding was increased slightly as a result of the cross-link. The corresponding reduced protein was significantly less stable than the wild type. Unfolding and refolding kinetics were carried out in 35% methanol, pH 6.8 at -15 degrees C, with guanidine hydrochloride as the denaturant. Unfolding/refolding of the wild-type and reduced enzyme showed biphasic kinetics both within and outside the denaturant-induced transition region and were consistent with the presence of a populated intermediate in folding. Double-jump refolding experiments eliminated proline isomerization as a possible cause for the biphasicity. The disulfide mutant protein, however, showed monophasic kinetics in all guanidine concentrations studied.     

Subunit interface of triosephosphate isomerase: site-directed mutagenesis and characterization of the altered enzyme

We have replaced asparagine residues at the subunit interface of yeast triosephosphate isomerase (TIM) using site-directed mutagenesis in order to elucidate the effects of substitutions on the catalytic activity and conformational stability of the enzyme. The mutant proteins were expressed in a strain of Escherichia coli lacking the bacterial isomerase and purified by ion-exchange and immunoadsorption chromatography. Single replacements of Asn-78 by either Thr or Ile residues had little effect on the enzyme's catalytic efficiency, while the single replacement Asn-78----Asp-78 and the double replacement Asn-14/Asn-78----Thr-14/Ile-78 appreciably lowered kcat for the substrate D-glyceraldehyde 3-phosphate. The isoelectric point of the mutant Asn-78----Asp-78 was equivalent to that of wild-type yeast TIM that had undergone a single, heat-induced deamidation, and this mutant enzyme was less resistant than wild-type TIM to denaturation and inactivation caused by elevated temperature, denaturants, tetrabutylammonium bromide, alkaline pH, and proteases.     

Structural studies of mutants of T4 lysozyme that alter hydrophobic stabilization

Multiple replacements at amino acid position 3 of bacteriophage T4 lysozyme have shown that the conformational stability of the protein is directly governed by the hydrophobicity of the residue substituted (Matsumura, M., Becktel, W. J., and Matthews, B. W. (1988) Nature 334, 406-410). Of the 13 mutant lysozymes made by site-directed mutagenesis, two variants, one with valine (I3V) and the other with tyrosine (I3Y), were crystallized and their structures solved. In this report we describe the crystal structures of these variants at 1.7 A resolution. While the structure of the I3V mutant is essentially the same as that of wild-type lysozyme, the I3Y mutant has substantial changes in its structure. The most significant of these are that the side chain of the tyrosine is not accommodated within the interior of the protein and the amino-terminal polypeptide (residues 1-9) moves 0.6-1.1 A relative to the wild-type structure. Using coordinates based on the wild-type and available mutant structures, solvent accessible surface area of residue 3 as well as the adjacent 9 residues in the folded form were calculated. The free energy of stabilization based on the transfer of these residues from a fully extended form to the interior to the folded protein was found to correlate well with the protein stability determined by thermodynamic analysis. The enhanced thermostability of the variant Ile-3----Leu, relative to wild-type lysozyme, can also be rationalized by surface-area calculations based on a model-built structure. Noncrystallization of most lysozyme variants at position 3 appears to be due to disruption of intermolecular contacts in the crystal. The Ile-3----Val variant is closely isomorphous with wild-type and maintains the same crystal contacts. In the Ile-3----Tyr variant, however, a new set of contacts is made in which direct protein-protein hydrogen bonds are replaced by protein-water-protein hydrogen bonds as well as a novel hydrogen bond involving the phenolic hydroxyl of the substituted tyrosine.     

Structural and thermodynamic analysis of compensating mutations within the core of chicken egg white lysozyme.

High resolution crystal structures have been determined for six chicken-type lysozymes that were constructed to investigate putative intermediates in the evolution of the lysozymes of modern game birds (Malcolm, B. A., Wilson, K. P., Matthews, B. W., Kirsch, J. F., and Wilson, A. C. (1990) Nature 345, 86-89). The amino acid replacements include Thr-40----Ser, Ile-55----Val, and Ser-91----Thr, as well as combinations of these substitutions. Residues 40, 55, and 91 are buried within the core of chicken lysozyme. The replacements therefore involve the insertion and/or removal of methyl groups from the protein interior. The mutant proteins have normal activities, and their thermal stabilities span a range of 7 degrees C, with some variants more stable and some less stable than the naturally occurring forms. Comparison of the crystal structures shows the overall structures to be very similar, but there are differences in the packing of side chains in the region of the replacements. The x-ray coordinates were used to evaluate the repacking of side chains in the protein interior and to attempt to evaluate the contributions of the different energetic interactions toward the overall stability of each variant. The results illustrate how proteins can compensate for potentially destabilizing substitutions in different ways and underscore the importance of high resolution structural data if changes in protein thermostability due to changes in protein sequence are to be understood. The findings also suggest that protein stability can be increased by mutations that lower strain in the protein interior while maintaining total buried hydrophobic surface area.     

Critical residues for structure and catalysis in short-chain dehydrogenases/reductases

Short-chain dehydrogenases/reductases form a large, evolutionarily old family of NAD(P)(H)-dependent enzymes with over 60 genes found in the human genome. Despite low levels of sequence identity (often 10-30%), the three-dimensional structures display a highly similar alpha/beta folding pattern. We have analyzed the role of several conserved residues regarding folding, stability, steady-state kinetics, and coenzyme binding using bacterial 3beta/17beta-hydroxysteroid dehydrogenase and selected mutants. Structure determination of the wild-type enzyme at 1.2-A resolution by x-ray crystallography and docking analysis was used to interpret the biochemical data. Enzyme kinetic data from mutagenetic replacements emphasize the critical role of residues Thr-12, Asp-60, Asn-86, Asn-87, and Ala-88 in coenzyme binding and catalysis. The data also demonstrate essential interactions of Asn-111 with active site residues. A general role of its side chain interactions for maintenance of the active site configuration to build up a proton relay system is proposed. This extends the previously recognized catalytic triad of Ser-Tyr-Lys residues to form a tetrad of Asn-Ser-Tyr-Lys in the majority of characterized short-chain dehydrogenases/reductase enzymes.     

Mutations in the autoinhibitor site of the regulatory subunit of cAMP-dependent protein kinase I. Replacement of Ala-97 and Ser-99 interferes with reassociation with the catalytic subunit.

Each regulatory (R) subunit of cAMP-dependent protein kinase contains an autoinhibitor site that lies approximately 90-100 residues from the amino terminus. In order to study the importance of this autoinhibitor site in the type I R-subunit for interacting with the catalytic (C) subunit, recombinant techniques were used to replace Ala-97 with Gln, His, Lys, and Arg and to replace Ser-99 with Gly and Lys. All of the mutant proteins having a replacement at Ala-97 showed reduced affinity for the C-subunit ranging from 14- to 55-fold. In general, the decrease in affinity of the Ala-97 mutants for the C-subunit correlated with the increase in size of the side chain. In contrast to wild type R-subunit, where MgATP facilitates holoenzyme formation, MgATP inhibits the reassociation in all of the Ala-97 mutants suggesting that the larger side chains sterically interfere with bound MgATP in the active site of the C-subunit. Whereas MgATP slowed holoenzyme formation, AMP actually accelerated the reassociation of the A97K, A97H (pH 6.0), and A97Q mutants with the C-subunit. Therefore, the side chains of Lys-97, His-97, and Gln-97 can interact either electrostatically or by hydrogen bonding with the phosphate of AMP. This interpretation is reinforced by the fact that the stimulatory effect of AMP on the A97H mutant was pH-dependent. The affinities of the S99G and S99K mutants for the C-subunit were reduced 7- and 24-fold, respectively, suggesting that Ser-99 also may contribute to interactions between the R- and C-subunits.     

Primary structure of a 21-kD protein from potato tubers

A 21-kD protein isolated earlier from potato tubers (Solanum tuberosum L.) has two isoforms, with pI 6.3 and 5.2, which were separated by fast protein ion-exchange chromatography on a Mono Q column. The primary structures of the two forms consisted of 187 and 186 amino acid residues. Both isoforms are composed of two polypeptide chains, designated A and B, linked by a single disulfide bond between Cys-146 of the A chain and Cys-7 of the B chain. The amino acid sequences of the A chains of the two forms, consisting of 150 residues each, differ in a single amino acid residue at position 52 (Val --> Ile), while the B chains, containing 37 and 36 residues, respectively, have substitutions at nine positions (Leu-8 --> Ser-8, Lys-25--Asp-26 --> Asn-25--Glu-26, Ile-31--Ser-32 --> Val-31--Leu-32, Lys-34--Gln-35--Val-36--Gln-37 --> Gln-34--Glu-35--Val-36). Both isoforms form stable inhibiting complexes with human leukocyte elastase and are less effective against chymotrypsin and trypsin.     

Fine structure-activity analysis of mutations at position 51 of tyrosyl-tRNA synthetase

Residue Thr-51 at the active site of tyrosyl-tRNA synthetase (Bacillus stearothermophilus) has been replaced with all the smaller amino acids by protein engineering to investigate direct and indirect effects of mutation on substrate binding and catalysis. The gamma-hydroxyl group of Thr-51 was thought to be 0.5 A too far from the ribose ring oxygen of ATP to form a hydrogen bond. Consistent with this, it is found that mutation of Thr-51----Cys-51, which should place the gamma-thiol group within its correct distance for hydrogen bonding, increases the affinity of the enzyme for ATP. Other mutations (Ser-51, Ala-51, and Gly-51) show the contributions to binding of the other atoms in the side chain of Thr-51. A family of enzymes has been produced, TyrTS(Thr-51) (wild type), TyrTS(Ala-51), TyrTS(Cys-51), and TyrTS(Pro-51), in which the value of kcat/KM for ATP in aminoacylation increases along the series. This is achieved by the value of KM decreasing significantly (2.5, 1.25, 0.29, and 0.019 mM, respectively) while there are smaller decreases in kcat (4.7, 4.0, 2.9, and 1.8 s-1, respectively). These variations cause each one of the enzymes to be more active than the others at particular concentrations of ATP. For example, at concentrations of ATP greater than 5.9 mM, TyrTS(Thr-51) is the most active, while TyrTS(Ala-51), TyrTS(Cys-51), and TyrTS(Pro-51) are the most active at 5.9-2.2, 2.2-0.42, and less than 0.42 mM ATP, respectively. Interestingly, position 51 shows variation in tyrosyl-tRNA synthetases isolated from different organisms.     

The importance of Asn47 for structure and reactivity of azurin from Alcaligenes denitrificans as studied by site-directed mutagenesis and spectroscopy.

To study the importance of a rigid copper site for the structure and function of azurin, a mutant with a reduced number of internal hydrogen bonds around the copper has been prepared and characterized. To this purpose, the previously cloned azu gene from Alcaligenes denitrificans (Hoitink, C. W. G., Woudt, L. P., Turenhout, J. C. M., Van de Kamp, M., and Canters, G. W. (1990) Gene (Amst.) 90, 15-20) was expressed in Escherichia coli and an isolation and purification procedure for the azurin was developed. The azurin obtained after heterologous expression in E. coli appears spectroscopically indistinguishable from azurin derived from A. denitrificans. The hydrogen bonding network around the copper site was altered by replacing Asn47 by a leucine by means of site-directed mutagenesis. Asn47 is a conserved residue in all blue copper proteins of which the primary structure has been reported. Characterization of the mutant protein with UV-visible, electron spin resonance, and NMR spectroscopy, and comparison with the wild type azurin revealed that the structure of the copper site as well as the overall structure of the protein have been largely retained. The redox activity as measured by the electron self-exchange rate appears not to have changed either. However, the mutant differs from the wild type azurin with respect to stability and midpoint potential. Midpoint potentials of mutant and wild type azurin amount to 396 and 286 mV, respectively. The difference is due to sizable entropic and enthalpic contributions which to a large extent cancel. Possible explanations for the outcome of these experiments are discussed.     

Significance of local electrostatic interactions in staphylococcal nuclease studied by site-directed mutagenesis

In this paper, we show that amino acids Glu(73) and Asp(77) of staphylococcal nuclease cooperate unequally with Glu(75) to stabilize its structure located between the C-terminal helix and beta-barrel of the protein. Amino acid substitutions E73G and D77G cause losses of the catalytic efficiency of 24 and 16% and cause thermal stability losses of 22 and 26%, respectively, in comparison with the wild type (WT) protein. However, these changes do not significantly change global and local secondary structures, based on measurements of fluorescence and CD(222 nm). Furthermore, x-ray diffraction analysis of the E75G protein shows that the overall structure of mutant and WT proteins is similar. However, this mutation does cause a loss of essential hydrogen bonding and charge interactions between Glu(75) and Lys(9), Tyr(93), and His(121). In experiments using double point mutations, E73G/D77G, E73G/E75G, and E75G/D77G, significant changes are seen in all mutants in comparison with WT protein as measured by fluorescence and CD spectroscopy. The losses of thermal stability are 47, 59, and 58%, for E73G/D77G, E73G/E75G, and E75G/D77G, respectively. The triple mutant, E73G/E75G/D77G, results in fluorescence intensity and CD(222 nm) close to those of the denatured state and in a thermal stability loss of 65% relative to the WT protein. Based on these results, we propose a model in which significant electrostatic interactions result in the formation of a locally stable structure in staphylococcal nuclease.     

Structural and mechanistic changes along an engineered path from metallo to nonmetallo 3-deoxy-D-manno-octulosonate 8-phosphate synthases

There are two classes of KDO8P synthases characterized respectively by the presence or absence of a metal in the active site. The nonmetallo KDO8PS from Escherichia coli and the metallo KDO8PS from Aquifex aeolicus are the best characterized members of each class. All amino acid residues that make important contacts with the substrates are conserved in both enzymes with the exception of Pro-10, Cys-11, Ser-235, and Gln-237 of the A. aeolicus enzyme, which correspond respectively to Met-25, Asn-26, Pro-252, and Ala-254 in the E. coli enzyme. Interconversion between the two forms of KDO8P synthases can be achieved by substituting the metal-coordinating cysteine of metallo synthases with the corresponding asparagine of nonmetallo synthases, and vice versa. In this report we describe the structural changes elicited by the C11N mutation and by three combinations of mutations (P10M/C11N, C11N/S235P/Q237A, and P10M/C11N/S235P/Q237A) situated along possible evolutionary paths connecting the A. aeolicus and the E. coli enzyme. All four mutants are not capable of binding metal and lack the structural asymmetry among subunits with regard to substrate binding and conformation of the L7 loop, which is typical of A. aeolicus wild-type KDO8PS but is absent in the E. coli enzyme. Despite the lack of the active site metal, the mutant enzymes display levels of activity ranging from 46% to 24% of the wild type. With the sole exception of the quadruple mutant, metal loss does not affect the thermal stability of KDO8PS. The free energy of unfolding in water is also either unchanged or even increased in the mutant enzymes, suggesting that the primary role of the active site metal in A. aeolicus KDO8PS is not to increase the enzyme stability. In all four mutants A5P binding displaces a water molecule located on the si side of PEP. In particular, in the double and triple mutant, A5P binds with the aldehyde carbonyl in hydrogen bond distance of Asn-11, while in the wild type this functional group points away from Cys-11. This alternative conformation of A5P is likely to have functional significance as it resembles the conformation of the acyclic reaction intermediate, which is observed here for the first time in some of the active sites of the triple mutant. The direct visualization of this intermediate by X-ray crystallography confirms earlier mechanistic models of KDO8P synthesis. In particular, the configuration of the C2 chiral center of the intermediate supports a model of the reaction in nonmetallo KDO8PS, in which water attacks an oxocarbenium ion or PEP from the si side of C2. Several explanations are offered to reconcile this observation with the fact that no water molecule is observed at this position in the mutant enzymes in the presence of both PEP and A5P. Significant differences were observed between the wild-type and the mutant enzymes in the Km values for PEP and A5P and in the Kd values for inorganic phosphate and R5P. These differences may reflect an evolutionary adaptation of metallo and nonmetallo KDO8PS's to the cellular concentrations of these metabolites in their respective hosts.     

Toward engineering the stability and hemin-binding properties of microsomal cytochromes b5 into rat outer mitochondrial membrane cytochrome b5: examining the influence of residues 25 and 71

As part of a larger effort to engineer the stability and hemin-binding properties of microsomal (Mc) cytochromes b(5) into rat liver outer mitochondrial membrane (OM) cytochrome (cyt) b(5), several mutants of rat OM cyt b(5) were prepared to study the effect of gradual and complete elimination of two extended hydrophobic networks, which are present in the structure of the mitochondrial protein and are absent in the structure of mammalian Mc cytochromes b(5). One of the hydrophobic networks, identified in a previous study [Altuve, A., Silchenko, S., Lee, K.-H., Kuczera, K., Terzyan, S., Zhang, X., Benson, D. R., and Rivera, M. (2001) Biochemistry 40, 9469-9483], encompasses the side chains of Ala-18, Ile-32, Leu-36, and Leu-47, whereas a second hydrophobic network, identified as part of this work, encompasses the side chains of Ile-25, Phe-58, Leu-71, and the heme. The X-ray structure of the A18S/I25L/I32L/L47R/L71S quintuple mutant of rat OM cyt b(5) demonstrates that both hydrophobic networks have been eliminated and that the corresponding structural elements of the Mc isoform have been introduced. The stability of the rat OM mutant proteins studied was found to decrease in the order wild type > I25L > A18S/I32L/L47R > L71S > A18S/I32L/L47R/L71S > 18S/I25L/I32L/L47R/L71S, indicating that the two hydrophobic networks do indeed contribute to the high stability of rat OM cyt b(5) relative to the bovine Mc isoform. Surprisingly, the quintuple mutant of rat OM cyt b(5) is less stable than bovine Mc cyt b(5), even though the former exhibits significantly slower rates of hemin release and hemin reorientation at pH 7.0. However, at pH 5.0 the bovine Mc and rat OM quintuple mutant proteins release hemin at comparable rates, suggesting that one or both of the His axial ligands in the rat OM protein are more resistant to protonation under physiological conditions. Results obtained from chemical denaturation experiments conducted with the apoproteins demonstrated that mutants containing L71S are significantly less stable than bovine Mc apocyt b(5), strongly suggesting that Leu-71 plays a pivotal role in the stabilization of rat OM apocyt b(5), presumably via hydrophobic interactions with Ile-25 and Phe-58. Because comparable interactions are absent in bovine Mc apocyt b(5), which contains Ser at position 71, it must resort to different interactions to stabilize its fold, thus highlighting yet another difference between rat OM and bovine Mc cyt b(5). During the course of these investigations we also discovered that rat OM cyt b(5) can be made to strongly favor hemin orientational isomer A (I32L) or isomer B (L71S) with a single point mutation and that release of hemin orientational isomers A and B can be kinetically resolved in certain rat OM mutants.     

Human cationic trypsinogen. Role of Asn-21 in zymogen activation and implications in hereditary pancreatitis

Mutation Asn-21 --> Ile in human cationic trypsinogen (Tg-1) has been associated with hereditary pancreatitis. Recent studies with rat anionic Tg (Tg-2) indicated that the analogous Thr-21 --> Ile mutation stabilizes the zymogen against autoactivation, whereas it has no effect on catalytic properties or autolytic stability of trypsin (Sahin-Tóth, M. (1999) J. Biol. Chem. 274, 29699-29704). In the present paper, human cationic Tg (Asn-21-Tg) and mutants Asn-21 --> Ile (Ile-21-Tg) and Asn-21 --> Thr (Thr-21-Tg) were expressed in Escherichia coli, and zymogen activation, zymogen degradation, and trypsin autolysis were studied. Enterokinase activated Asn-21-Tg approximately 2-fold better than Ile-21-Tg or Thr-21-Tg, and catalytic parameters of trypsins were comparable. At 37 degrees C, in 5 mm Ca(2+), all three trypsins were highly stable. In the absence of Ca(2+), Asn-21- and Ile-21-trypsins suffered autolysis in an indistinguishable manner, whereas Thr-21-trypsin exhibited significantly increased stability. In sharp contrast to observations with the rat proenzyme, at pH 8.0, 37 degrees C, autoactivation kinetics of Asn-21-Tg and Ile-21-Tg were identical; however, at pH 5. 0, Ile-21-Tg autoactivated at an enhanced rate relative to Asn-21-Tg. Remarkably, at both pH values, Thr-21-Tg showed markedly higher autoactivation rates than the two other zymogens. Finally, autocatalytic proteolysis of human zymogens was limited to cleavage at Arg-117, and no digestion at Lys-188 was detected. The observations indicate that zymogen stabilization by Ile-21 as observed in rat Tg-2 is not characteristic of human Tg-1. Instead, an increased propensity to autoactivation under acidic conditions might be relevant to the pathomechanism of the Asn-21 --> Ile mutation in hereditary pancreatitis. In the same context, faster autoactivation and increased trypsin stability caused by the Asn-21 --> Thr mutation in human Tg-1 might provide a rationale for the evolutionary divergence from Thr-21 found in other mammalian trypsinogens.     

Site-directed mutageneses of rat liver cytochrome P-450d: axial ligand and heme incorporation

By oligonucleotide-directed mutageneses, 13 substitutions of amino acids at the carboxy-terminal region of rat liver cytochrome P-450d were done as follows: (A) Phe-449----Tyr; (B) Gly-450----Ser; (C) Leu-451----Ser; (D) Gly-452----Glu; (E) Lys-453----Glu; (F) Arg-454----Leu; (G) Arg-455----Gly; (H) Cys-456----Tyr; (I) Cys-456----His; (J) Ile-457----Ser; (K) Gly-458----Glu; (L) Glu-459----Ala; (M) Ile-460----Ser. The CO-bound reduced forms of the wild type and mutants B-G, J, L, and M gave Soret peaks at 448 nm. The CO complex of mutant A gave a Soret peak at 445 nm. The intensities of the CO-bound forms of mutants A, C, D, and J were very small compared with that of the wild-type complex. The CO-reduced forms of mutants H, I, and K did not give a Soret peak around 450 nm at all. The 448-nm peak of mutant F was unstable and quickly disappeared with the concomitant appearance of a peak at 420 nm.(ABSTRACT TRUNCATED AT 250 WORDS)