Helix formation in enzymically ligated peptides as a driving force for the synthetic reaction: example of alpha-globin semisynthetic reaction.

The alpha-globin semisynthetic reaction, namely, the ligation of the complementary fragments of alpha-globin, alpha 1-30 and alpha 31-141, in the presence of 30% l-propanol that is catalyzed by V8 protease is distinct as compared with the previously studied protease-catalyzed splicing of the discontinuity sites of the fragment complementing systems [Sahni et al. (1989) Biochemistry 28, 5456]. The complementary fragments of alpha-globin do not exhibit noncovalent interaction between them even in the presence of l-propanol, the organic cosolvent used to facilitate the alpha-globin semisynthetic reaction. Besides, a significant portion of the fragment alpha 31-141 does not contribute to the protease-catalyzed splicing reaction. Alpha 1-30 and alpha 31-40 are ligated by V8 protease to yield alpha 1-40 in much the same way as the splicing of alpha 1-30 with either alpha 31-141 or alpha 31-47 to yield alpha-globin or alpha 1-47, respectively. An equimolar mixture of alpha 1-30 and alpha 31-40 does not show any 'complexation' in the presence of 30% l-propanol, the medium used for the synthetic reaction. The splicing junction, i.e., Glu30-Arg31 peptide bond, is located in the middle of the B-helix (residues 20-35) of the parent protein. Most of the residues from the A-helix of the protein could also be deleted from segment alpha 1-30 without influencing the V8 protease-catalyzed splicing reaction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthetic potential of Staphylococcus aureus V8-protease: an approach toward semisynthesis of covalent analogs of alpha-chain of hemoglobin S

Enzyme-catalyzed reformation of peptide bonds in the noncovalent fragment systems of proteins has been emerging as a convenient procedure for the semisynthesis of covalent analogs of the respective proteins. Limited proteolysis of the alpha-chain of hemoglobin S with Staphylococcus aureus V8-protease converts the chain into a fragment-complementing system by hydrolyzing the peptide bond Glu(30)-Arg(31) of the chain. Therefore, it is conceivable that semisynthesis of covalent analogs of alpha-chain could be achieved if conditions for the V8-protease catalyzed formation of peptide bonds could be established. The synthetic potential of V8-protease has been now investigated by incubating V8-protease-derived fragments of alpha-chain, namely alpha 1-30 and alpha 31-47 with the enzyme at pH 6.0 in the presence of n-propanol as the organic cosolvent. RP high performance liquid chromatography analysis showed that a new chromatographically distinct component is generated on incubation, and this has been identified as alpha 1-47 by amino acid analysis, redigestion with V8-protease (in the absence of n-propanol), and tryptic peptide mapping. Optimal conditions for the synthesis of alpha 1-47 is at pH 6.0, 4 degrees C, and 24 hr of incubation with 25% n-propanol as organic cosolvent. This stereospecific condensation of the fragments proceeded to a high level of about 50% in 24 hr. Further incubation up to 72 hr did not increase the yield of alpha 1-47, suggesting that an equilibration of synthesis and hydrolysis reactions has been attained. The demonstration of the synthetic potential of V8-protease and the fact that alpha 1-30 and alpha 31-141 interact to form a native-like complex, opens up an approach for the semisynthesis of covalent analogs of alpha-chain of hemoglobin S.     

Permissible discontinuity region of the alpha-chain of hemoglobin: noncovalent interaction of heme and the complementary fragments alpha 1-30 and alpha 31-141

Generation of a fragment-complementing system of the alpha-chain on limited proteolysis with Staphylococcus aureus V8 protease has been investigated. Digestion of the alpha-chain (0.4 mM) of hemoglobin with V8 protease in phosphate buffer at pH 6.0 and 37 degrees C is limited to the peptide bonds of Glu-23, Glu-27, Glu-30, and Asp-47. Gel filtration of a V8 protease digest of the alpha-chain on a Sephadex G-50 column did not release any heme to the low molecular weight region, though some peptides were released from the protein. The filtration studies revealed the presence of two heme-containing components in the digest, the major one eluting at the alpha-chain position and the minor one eluting slightly ahead of the alpha-chain position. Reversed-phase high-performance liquid chromatography and amino-terminal sequence analysis demonstrated that the component eluting at the alpha-chain position contains species generated by the noncovalent interactions of heme and the complementary fragments alpha 1-30 and alpha 31-141. In dilute solutions (0.04 mM) the V8 protease digestion occurred exclusively on the carboxyl side of Glu-30(alpha). This high selectivity was also observed at pH 4.0 and pH 7.8. The visible spectra and the ultraviolet circular dichroic spectra of the digest reflect the native-like structure of the noncovalent fragment system. The dissociation constant of alpha 1-30 appears to be in the range of 10(-8) M. In tetrameric hemoglobin A the peptide bond of Glu-30-Arg-31 of the alpha-chain is not accessible to V8 protease digestion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Semisynthetic hemoglobin A: reconstitution of functional tetramer from semisynthetic alpha-globin

The optimal conditions for the semisynthesis of alpha-globin through Staphylococcus aureus V8 protease condensation of a synthetic fragment (alpha 1-30) with the complementary apo fragment (alpha 31-141) in the presence of structure-inducing organic cosolvents and the reconstitution of the functional tetramer from semisynthetic alpha-globin have been investigated. The protease-catalyzed ligation of the complementary apo fragments alpha 1-30 and alpha 31-141 proceeds with very high selectivity at pH 6.0 and 4 degrees C in the presence of 1-propanol as the organic cosolvent. A 30% 1-propanol solution was optimal for the semisynthetic reaction, and the synthetic reaction attained an equilibrium (approximately 50%) in 72 h. The synthetic reaction proceeds smoothly over a wide pH range (pH 5-8). Besides, the semisynthetic system is flexible, and it also proceeded well if trifluoroethanol or 2-propanol was used instead of 1-propanol. However, glycerol, a versatile organic cosolvent used in all other proteosynthetic reactions reported in the literature, was not very efficient as an organic cosolvent in the present synthetic reaction. The semisynthetic alpha-globin prepared with 1-propanol as the organic cosolvent has been reconstituted into HbA. The semisynthetic HbA was then purified by CM-cellulose chromatography. The semisynthetic HbA is indistinguishable from native HbA, in terms of its structural and functional properties. The semisynthetic approach provides the flexibility in protein engineering studies for the incorporation of spectroscopic labels (13C- and/or 15N-labeled amino acids), noncoded amino acids, or unnatural bond functionalities, which at present is not possible with genetic approaches.     

Inhibition of beta(S)-chain dependent polymerization by synergistic complementation of contact site perturbations of alpha-chain: application of semisynthetic chimeric alpha-chains

Mouse alpha(1-30)-horse alpha(31-141) chimeric alpha-chain, a semisynthetic super-inhibitory alpha-chain, inhibits beta(S)-chain dependent polymerization better than both parent alpha-chains. Although contact site sequence differences are absent in the alpha(1-30) region of the chimeric chain, the four sequence differences of the region alpha(17-22) could induce perturbations of the side chains at alpha(16), alpha(20) and alpha(23), the three contact sites of the region. A synergistic complementation of such contact site perturbation with that of horse alpha(31-141) probably results in the super-inhibitory activity of the chimeric alpha-chain. The inhibitory contact site sequence differences, by themselves, could also exhibit similar synergistic complementation. Accordingly, the polymerization inhibitory activity of Hb Le-Lamentin (LM) mutation [His20(alpha)-->Gln], a contact site sequence difference, engineered into human-horse chimeric alpha-chain has been investigated to map such a synergistic complementation. Gln20(alpha) has little effect on the O(2) affinity of HbS, but in human-horse chimeric alpha-chain it reduces the O(2) affinity slightly. In the chimeric alpha-chain, Gln20(alpha) increased sensitivity of the betabeta cleft for the DPG influence, reflecting a cross-talk between the alpha(1)beta(1) interface and betabeta cleft in this semisynthetic chimeric HbS. In the human alpha-chain frame, the polymerization inhibitory activity of Gln20(alpha) is higher compared with horse alpha(1-30), but lower than mouse alpha(1-30). Gln20(alpha) synergistically complements the inhibitory propensity of horse alpha(31-141). However, the inhibitory activity of LM-horse chimeric alpha-chain is still lower than that of mouse-horse chimeric alpha-chain. Therefore, perturbation of multiple contact sites in the alpha(1-30) region of the mouse-horse chimeric alpha-chain and its linkage with the inhibitory propensity of horse alpha(31-141) has been now invoked to explain the super-inhibitory activity of the chimeric alpha-chain. The 'linkage-map' of contact sites can serve as a blueprint for designing synergistic complementation of multiple contact sites into alpha-chains as a strategy for generating super-inhibitory antisickling hemoglobins for gene therapy of sickle cell disease.     

Interspecies hybrid HbS: complete neutralization of Val6(beta)-dependent polymerization of human beta-chain by pig alpha-chains

Interspecies hybrid HbS (alpha(2)(P)beta(2)(S)), has been assembled in vitro from pig alpha-globin and human beta(S)-chain. The alpha(2)(P)beta(2)(S) retains normal tetrameric structure (alpha(2)beta(2)) of human Hb and an O(2) affinity comparable to that of HbS in 50 mM Hepes buffer; but, its O(2) affinity is slightly higher than that of HbS in the presence of allosteric effectors (chloride, DPG and phosphate). The (1)H-NMR spectroscopy detected distinct differences between the heme environments and alpha(1)beta(1) interfaces of pig Hb and HbS, while their alpha(1)beta(2) interfaces appear very similar. The interspecies hybrid alpha(2)(H)beta(2)(P) resembles pig Hb; the pig beta-chain dictated the conformation of the heme environment of the human alpha-subunit, and to the alpha(1)beta(1) interfaces of the hybrid. In the alpha(2)(P)beta(2)(S) hybrid, beta(S)-chain dictated the conformation of human heme environment to the pig alpha-chain in the hybrid; but the conformation of alpha(1)beta(1) interface of this hybrid is close to, but not identical to that of HbS. On the other hand, the alpha(1)beta(2) interface conformation is identical to that of HbS. More important, the alpha(2)(P)beta(2)(S) does not polymerize when deoxygenated; pig alpha-chain completely neutralizes the beta(S)-chain dependent polymerization. The polymerization inhibitory propensity of pig alpha-chain is higher when it is present in the cis alpha(P)beta(S) dimer relative to that in a trans alpha(P)beta(A) dimer. The semisynthetically generated chimeric pig-human and human-pig alpha-chains by exchanging the alpha(1-30) segments of human and pig alpha-chains have established that the sequence differences of pig alpha(31-141) segment can also completely neutralize the polymerization. Comparison of the electrostatic potential energy landscape of the alpha-chain surfaces of HbS and alpha(2)(P)beta(2)(S) suggests that the differences in electrostatic potential energy surfaces on the alpha-chain of alpha(2)(P)beta(2)(S) relative to that in HbS, particularly the ones involving CD region, E-helix and EF-corner of pig alpha-chain are responsible for the polymerization neutralization activity. The pig and human-pig chimeric alpha-chains can serve as blueprints for the design of a new generation of variants of alpha-chain(s) suitable for the gene therapy of sickle cell disease.     

Subunit structured of pig small-intestinal brush-border aminopeptidase N.

Aminopeptidase N (EC 3.4.11.2), when isolated from pig intestine in either the proteinase- or detergent-released form, frequently appears to contain three polypeptide chains, here termed alpha, beta and gamma. We have established by an immunological technique that the beta- and gamma-polypeptides are derived from the alpha-chain and that the intact enzyme is a dimer, alpha 2. Each alpha-chain of the detergent form was shown to contain a hydrophobic anchor peptide about 35 amino acid residues in length, which included the N-terminal sequences. A peptide bond in the alpha-chain was very sensitive to proteolysis. Its cleavage generated the commonly observed forms: alpha beta gamma and beta 2 gamma 2. The gamma-fragment, which lacked the anchor peptide, was derived from the C-terminal part of the alpha-chain.     

Engineering subtilisin E for enhanced stability and activity in polar organic solvents

We examined the effect of a novel disulfide bond engineered in subtilisin E from Bacillus subtilis based on the structure of a thermophilic subtilisin-type serine protease aqualysin I. Four sites (Ser163/Ser194, Lys170/Ser194, Lys170/Glu195, and Pro172/Glu195) in subtilisin E were chosen as candidates for Cys substitutions by site-directed mutagenesis. The Cys170/Cys195 mutant subtilisin formed a disulfide bond in B. subtilis, and showed a 5-10-fold increase in specific activity for an authentic peptide substrate for subtilisin, N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide, compared with the single-Cys mutants. However, the disulfide mutant had a 50% decrease in catalytic efficiency due to a smaller k(cat) and was thermolabile relative to the wild-type enzyme, whereas it was greatly stabilized relative to its reduced form. These results suggest that an electrostatic interaction between Lys170 and Glu195 is important for catalysis and stability in subtilisin E. Interestingly, the disulfide mutant was found to be more stable in polar organic solvents, such as dimethylformamide and ethanol, than the wild-type enzyme, even under reducing conditions; this is probably due to the substitution of uncharged Cys by charged surface residues (Lys170 and Glu195). Further, the amino-terminal engineered disulfide bond (Gly61Cys/Ser98Cys) and the mutation Ile31Leu were introduced to enhance the stability and catalytic activity. A prominent 3-4-fold increase in the catalytic efficiency occurred in the quintet mutant enzyme over the range of dimethylformamide concentration (up to 40%).     

Three subunits contribute amino acids to the active site of tetrameric adenylosuccinate lyase: Lys268 and Glu275 are required

Tetrameric adenylosuccinate lyase (ASL) of Bacillus subtilis catalyzes the cleavage of adenylosuccinate to form AMP and fumarate. We previously reported that two distinct subunits contribute residues to each active site, including the His68 and His89 from one and His141 from a second subunit [Brosius, J. L., and Colman, R. F. (2000) Biochemistry 39, 13336-13343]. Glu(275) is 2.8 A from His141 in the ASL crystal structure, and Lys268 is also in the active site region; Glu275 and Lys268 come from a third, distinct subunit. Using site-directed mutagenesis, we have replaced Lys268 by Arg, Gln, Glu, and Ala, with specific activities of the purified mutant enzymes being 0.055, 0.00069, 0.00028, and 0.0, respectively, compared to 1.56 units/mg for wild-type (WT) enzyme. Glu275 was substituted by Gln, Asp, Ala, and Arg; none of these homogeneous mutant enzymes has detectable activity. Circular dichroism and light scattering reveal that neither the secondary structure nor the oligomeric state of the Lys268 mutant enzymes has been perturbed. Native gel electrophoresis and circular dichroism indicate that the Glu275 mutant enzymes are tetramers, but their conformation is altered slightly. For K268R, the K(m)s for all substrates are similar to WT enzyme. Binding studies using [2-3H]-adenylosuccinate reveal that none of the Glu275 mutant enzymes, nor inactive K268A, can bind substrate. We propose that Lys268 participates in binding substrate and that Glu275 is essential for catalysis because of its interaction with His141. Incubation of H89Q with K268Q or E275Q leads to restoration of up to 16% WT activity, while incubation of H141Q with K268Q or E275Q results in 6% WT activity. These complementation studies provide the first functional evidence that a third subunit contributes residues to each intersubunit active site of ASL. Thus, adenylosuccinate lyase has four active sites per enzyme tetramer, each of which is formed from regions of three subunits.     

Functional role of the distal valine (E11) residue of alpha subunits in human haemoglobin

We have expressed human alpha-globin to a high level in Escherichia coli as a fusion protein, purified it and removed the N-terminal leader sequence by site-specific proteolysis with blood coagulation factor Xa. The apo globin has been refolded and reconstituted with haem and native beta-globin to form fully functional haemoglobin (Hb) with properties identical to those of native human Hb. By site-directed mutagenesis we have altered the distal residues of the alpha subunits and compared the functional properties of these mutant proteins. The rates of various ligands binding to these proteins in the R-state have been reported by Mathews et al. Here, we present the oxygen equilibrium curves of three E11 alpha mutants and the crystal structures of two of these mutants in the deoxy form. Replacing the distal valine residue of alpha-globin with alanine, leucine or isoleucine has no effect on the oxygen affinity of the protein in either quaternary state, in contrast to the equivalent mutations of beta subunits. The crystal structure of the valine E11 alpha----isoleucine mutant shows that the larger E11 residue excludes water from the haem pocket, but causes no significant movement of other amino acid residues. We conclude that the distal valine residue of alpha-globin does not control the oxygen affinity of the protein by sterically hindering ligand binding.     

A key role in catalysis for His89 of adenylosuccinate lyase of Bacillus subtilis

Adenylosuccinate lyase of Bacillus subtilis is a tetrameric enzyme which catalyzes the cleavage of adenylosuccinate to AMP and fumarate. We have mutated His(89), one of three conserved histidines, to Gln, Ala, Glu, and Arg. The enzymes were expressed in Escherichia coli and purified to homogeneity. As compared to a specific activity of 1. 56 micromol of adenylosuccinate converted/min/mg protein for wild-type enzyme, the mutant enzymes exhibit specific activities of 0.0225, 0.0036, 0.0036, and 0.0009 for H89Q, H89A, H89E, and H89R, respectively. Circular dichroism and FPLC gel filtration reveal that mutant enzymes have a similar conformation and oligomeric state to that of wild-type enzyme. In H89Q, the K(M) for adenylosuccinate increases slightly to 2.5-fold that of wild-type, the K(M) for fumarate is elevated 3.3-fold, and the K(M) for AMP is 13 times higher than that observed in wild-type enzyme. The catalytic efficiency of the H89Q enzyme is compromised, with k(cat)/K(M) reduced 174-fold in the direction of AMP formation. These data suggest that His(89) plays a role in both the binding of the AMP portion of the substrate and in correctly orienting the substrate for catalysis. Incubation of H89Q with inactive H141Q enzyme [Lee, T. T., Worby, C., Bao, Z.-Q., Dixon, J. E., and Colman, R. F. (1999) Biochemistry 38, 22-32] leads to a 30-fold increase in activity. This intersubunit complementation indicates that His(89) and His(141) from different subunits participate in the active site and that both are required for catalysis.     

Saturation mutagenesis reveals that GLU54 of norovirus 3C-like protease is not essential for the proteolytic activity

The norovirus 3C-like protease is a member of the chymotrypsin-like serine protease superfamily. Previous characterization of its crystal structure has implicated the Glu54-His30-Cys139 triad in the catalysis. In the present study, the Glu54 residue of the protease was subjected to site-saturation mutagenesis, with the result that nearly half of the mutants retained the significant proteolytic activity. It was suggested that a carboxylate at position 54 was not essential for the activity. The in vitro assays of the proteolysis revealed that most of Glu54 mutants retained relatively high proteolytic activity. When the Glu54 mutation was combined with the Ser mutation of the Cys139 residue, a nucleophile, only the Asp54 and Gln54 mutations showed proteolytic activity comparable to that of the Ser139 single mutant, suggesting that a hydrogen bond between Glu54 and His30 was critical in the Ser139 background. These results suggested that the mechanism of the proteolysis by the wild-type norovirus 3C-like protease was different from that of typical chymotrypsin-like serine proteases.     

X-ray crystallographic structure of the Norwalk virus protease at 1.5-A resolution

Norwalk virus (NV), a member of the Caliciviridae family, is the major cause of acute, epidemic, viral gastroenteritis. The NV genome is a positive sense, single-stranded RNA that encodes three open reading frames (ORFs). The first ORF produces a polyprotein that is processed by the viral cysteine protease into six nonstructural proteins. We have determined the structure of the NV protease to 1.5 and 2.2 A from crystals grown in the absence or presence, respectively, of the protease inhibitor AEBSF [4-(2-aminoethyl)-benzenesulfonyl fluoride]. The protease, which crystallizes as a stable dimer, exhibits a two-domain structure similar to those of other viral cysteine proteases with a catalytic triad composed of His 30, Glu 54, and Cys 139. The native structure of the protease reveals strong hydrogen bond interactions between His 30 and Glu 54, in the favorable syn configuration, indicating a role of Glu 54 during proteolysis. Mutation of this residue to Ala abolished the protease activity, in a fluorogenic peptide substrate assay, further substantiating the role of Glu 54 during proteolysis. These observations contrast with the suggestion, from a previous study of another norovirus protease, that this residue may not have a prominent role in proteolysis (K. Nakamura, Y. Someya, T. Kumasaka, G. Ueno, M. Yamamoto, T. Sato, N. Takeda, T. Miyamura, and N. Tanaka, J. Virol. 79:13685-13693, 2005). In the structure from crystals grown in the presence of AEBSF, Glu 54 undergoes a conformational change leading to disruption of the hydrogen bond interactions with His 30. Since AEBSF was not apparent in the electron density map, it is possible that these conformational changes are due to subtle changes in pH caused by its addition during crystallization.     

Glu88 in the non-catalytic domain of acylpeptide hydrolase plays dual roles: charge neutralization for enzymatic activity and formation of salt bridge for thermodynamic stability

Acylpeptide hydrolase of Aeropyrum pernix K1 is composed of a catalytic alpha/beta hydrolase domain and a non-catalytic beta-propeller domain. The Glu88 residue of the propeller domain is highly conserved in the prolyl oligopeptidase family and forms an inter-domain salt bridge with Arg526, a key residue for substrate binding. We have dissected the functions of Glu88 using site-directed mutagenesis, steady-state kinetics analyses, and molecular dynamics simulations. In E88A and E88A/R526K mutants, with a broken inter-domain salt bridge and a positive charge at position 526, catalytic activities for both a peptidase substrate and an esterase substrate were almost abolished. Analysis of the pH dependence of the mutants' reaction kinetics indicates that these mutations lead to changes in the electrostatic environment of the active site, which can be modulated by chloride ions. These findings indicate that the neutralization at position 526 is favorable for the activity of the enzyme, which is also verified by the catalytic behavior of E88A/R526V mutant. All mutants have lower thermodynamic stability than the wild-type. Therefore, Glu88 plays two major roles in the function of the enzyme: neutralizing the positive charge of Arg526, thereby increasing the enzymatic activity, and forming the Glu88-Arg526 salt bridge, thereby stabilizing the protein.     

Unusual proteolytic activation of pro-hepatocyte growth factor by plasma kallikrein and coagulation factor XIa

Hepatocyte growth factor (HGF), the ligand for the receptor tyrosine kinase c-Met, is composed of an alpha-chain containing four Kringle domains (K1-K4) and a serine protease domain-like beta-chain. Receptor activation by HGF is contingent upon prior proteolytic conversion of the secreted inactive single chain form (pro-HGF) into the biologically active two chain form by a single cleavage at the Arg(494)-Val(495) bond. By screening a panel of serine proteases we identified two new HGF activators, plasma kallikrein and coagulation factor XIa (FXIa). The concentrations of kallikrein and FXIa to cleave 50% (EC(50)) of (125)I-labeled pro-HGF during a 4-h period were 10 and 17 nm. Unlike other known activators, both FXIa and kallikrein processed pro-HGF by cleavage at two sites. Using N-terminal sequencing they were identified as the normal cleavage site Arg(494)-Val(495) and the novel site Arg(424)-His(425) located in the K4 domain of the alpha-chain. The identity of this unusual second cleavage site was firmly established by use of the double mutant HGF(R424A/R494E), which was completely resistant to cleavage by kallikrein and FXIa. Experiments with another mutant form, HGF(Arg(494) --> Glu), indicated that cleavage at the K4 site was independent of a prior cleavage at the primary, kinetically preferred Arg(494)-Val(495) site. The cleavage at the K4 site had no obvious consequences on HGF function, because it was fully capable of phosphorylating the c-Met receptor of A549 cells. This may be explained by the disulfide bond network in K4, which holds the cleaved alpha-chain together. In conclusion, the ability of plasma kallikrein and FXIa to activate pro-HGF in vitro raises the possibility that mediators of inflammation and blood coagulation may also regulate processes that involve the HGF/c-Met pathway, such as tissue repair and angiogenesis.     

Proteolysis of an active site peptide of lactate dehydrogenase by human immunodeficiency virus type 1 protease.

The muscle and heart lactate dehydrogenase (LDHs) of rabbit and pig are specifically cleaved at a single position by HIV-1 protease, resulting in the conversion of 36-kDa subunits of the oligomeric enzymes into 21- and 15-kDa protein bands as analyzed by SDS-PAGE. While the proteolysis was observed at neutral pH, it became more pronounced at pH 6.0 and 5.0. The time courses of the cleavage of the 36-kDa subunits were commensurate with the time-dependent loss of both quaternary structure and enzymatic activity. These results demonstrated that deoligomerization of rabbit muscle LDH at acidic pH rendered its subunits more susceptible to proteolysis, suggesting that a partially denatured form of the enzyme was the actual substrate. Proteolytic cleavage of the rabbit muscle enzyme occurred at a decapeptide sequence, His-Gly-Trp-Ile-Leu*Gly-Glu-His-Gly-Asp (scissile bond denoted throughout by an asterisk), which constitutes a "strand-loop" element in the muscle and heart LDH structures and contains the active site histidyl residue His-193. The kinetic parameters Km, Vmax/KmEt, and Vmax/Et for rabbit muscle LDH and the synthetic decapeptide Ac-His-Gly-Trp-Ile-Leu*Gly-Glu-His-Gly-Asp-NH2 were nearly identical, suggesting that the decapeptide within the protein substrate is conformationally mobile, as would be expected for the peptide substrate in solution. Insertion of part of this decapeptide sequence into bacterial galactokinase likewise rendered this protein susceptible to proteolysis by HIV-1 protease, and site-directed mutagenesis of this peptide in galactokinase revealed that the Glu residue at the P2' was important to binding to HIV-1 protease. Crystallographic analysis of HIV-1 protease complexed with a tight-binding peptide analogue inhibitor derived from this decapeptide sequence revealed that the "strand-loop" structure of the protein substrate must adopt a beta-sheet structure upon binding to the protease. The Glu residue in the P2' position of the inhibitor likely forms hydrogen-bonding interactions with both the alpha-amide and gamma-carboxylic groups of Asp-30 in the substrate binding site.     

Hb Catonsville (glutamic acid inserted between Pro-37(C2)alpha and Thr-38(C3)alpha). Nonallelic gene conversion in the globin system?

Hb Catonsville is an unstable variant in which glutamic acid is inserted into the alpha-globin chain between Pro-37(C2) and Thr-38(C3). The peptide sequence data are consistent with the DNA sequence of the polymerase chain reaction-amplified fragment of the variant globin gene, which shows the insertion of the triplet codon--GAA--into the mutant alpha-globin gene. In the normal alpha-globin gene cluster the codon for glutamic acid is GAG rather than GAA. Thus, there are two features unique to Hb Catonsville, one the insertion of a single residue into the interior of the alpha-globin chain, and two the presence of the alternate codon for glutamic acid. The experimental evidence suggests that Hb Catonsville may be an example of nonhomologous nonallelic gene conversion, an observation not previously reported in this gene family. The mutation occurs in the critical alpha 1 beta 2 interface of the hemoglobin tetramer and leads to a variant with high oxygen affinity, a reduced cooperativity, and Bohr effect.     

Alteration of sperm whale myoglobin heme axial ligation by site-directed mutagenesis

Three mutant proteins of sperm whale myoglobin (Mb) that exhibit altered axial ligations were constructed by site-directed mutagenesis of a synthetic gene for sperm whale myoglobin. Substitution of distal pocket residues, histidine E7 and valine E11, with tyrosine and glutamic acid generated His(E7)Tyr Mb and Val(E11)Glu Mb. The normal axial ligand residue, histidine F8, was also replaced with tyrosine, resulting in His(F8)Tyr Mb. These proteins are analogous in their substitutions to the naturally occurring hemoglobin M mutants (HbM). Tyrosine coordination to the ferric heme iron of His(E7)Tyr Mb and His(F8)Tyr Mb is suggested by optical absorption and EPR spectra and is verified by similarities to resonance Raman spectral bands assigned for iron-tyrosine proteins. His(E7)Tyr Mb is high-spin, six-coordinate with the ferric heme iron coordinated to the distal tyrosine and the proximal histidine, resembling Hb M Saskatoon [His(beta E7)Tyr], while the ferrous iron of this Mb mutant is high-spin, five-coordinate with ligation provided by the proximal histidine. His(F8)Tyr Mb is high-spin, five-coordinate in both the oxidized and reduced states, with the ferric heme iron liganded to the proximal tyrosine, resembling Hb M Iwate [His(alpha F8)Tyr] and Hb M Hyde Park [His(beta F8)Tyr]. Val(E11)Glu Mb is high-spin, six-coordinate with the ferric heme iron liganded to the F8 histidine. Glutamate coordination to the ferric iron of this mutant is strongly suggested by the optical and EPR spectral features, which are consistent with those observed for Hb M Milwaukee [Val(beta E11)Glu]. The ferrous iron of Val(E11)Glu Mb exhibits a five-coordinate structure with the F8 histidine-iron bond intact.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glutamic acid-141: a heme 'bodyguard' in anionic tobacco peroxidase

The role of the conserved glutamic acid residue in anionic plant peroxidases with regard to substrate specificity and stability was examined. A Glu141Phe substitution was generated in tobacco anionic peroxidase (TOP) to mimic neutral plant peroxidases such as horseradish peroxidase C (HRP C). The newly constructed enzyme was compared to wild-type recombinant TOP and HRP C expressed in E. coli. The Glu141Phe substitution supports heme entrapment during the refolding procedure and increases the reactivation yield to 30% compared to 7% for wild-type TOP. The mutation reduces the activity towards ABTS, o-phenylenediamine, guaiacol and ferrocyanide to 50% of the wild-type activity. No changes are observed with respect to activity for the lignin precursor substrates, coumaric and ferulic acid. The Glu141Phe mutation destabilizes the enzyme upon storage and against radical inactivation, mimicking inactivation in the reaction course. Structural alignment shows that Glu141 in TOP is likely to be hydrogen-bonded to Gln149, similar to the Glu143-Lys151 bond in Arabidopsis A2 peroxidase. Supposedly, the Glu141-Gln149 bond provides TOP with two different modes of stabilization: (1) it prevents heme dissociation, i.e., it 'guards' heme inside the active center; and (2) it constitutes a shield to protect the active center from solvent-derived radicals.     

Glutamate 47 in 1-aminocyclopropane-1-carboxylate synthase is a major specificity determinant

Glutamate 47 is conserved in 1-aminocyclopropane-1-carboxylate (ACC) synthases and is positioned near the sulfonium pole of (S,S)-S-adenosyl-L-methionine (SAM) in the modeled pyridoxal phosphate quinonoid complex with SAM. E47Q and E47D constructs of ACC synthase were made to investigate a putative ionic interaction between Glu47 and SAM. The k(cat)/K(m) values for the conversion of (S,S)-SAM to ACC and methylthioadenosine (MTA) are depressed 630- and 25-fold for the E47Q and E47D enzymes, respectively. The decreases in the specificity constants are due to reductions in k(cat) for both mutant enzymes, and a 5-fold increase in K(m) for the E47Q enzyme. Importantly, much smaller effects were observed for the kinetic parameters of reactions with the alternate substrates L-vinylglycine (L-VG) (deamination to form alpha-ketobutyrate and ammonia) and L-alanine (transamination to form pyruvate), which have uncharged side chains. L-VG is both a substrate and a mechanism-based inactivator of the enzyme [Feng, L., and Kirsch, J. F. (2000) Biochemistry 39, 2436-2444], but the partition ratio, k(cat)/k(inact), is unaffected by the Glu47 mutations. ACC synthase primarily catalyzes the beta,gamma-elimination of MTA from the (R,S) diastereomer of SAM to produce L-VG [Satoh, S., and Yang, S. F. (1989) Arch.Biochem. Biophys. 271, 107-112], but catalyzes the formation of ACC to a lesser extent via alpha,gamma-elimination of MTA. The partition ratios for (alpha,gamma/beta,gamma)-elimination on (R,S)-SAM are 0.4, < or =0.014, and < or =0.08 for the wild-type, E47Q, and E47D enzymes, respectively. The results of these experiments strongly support a role for Glu47 as an anchor for the sulfonium pole of (S,S)-SAM, and consequently a role as an active site determinant of reaction specificity.