Folding of homologous proteins: conservation of the folding mechanism of the alpha subunit of tryptophan synthase from Escherichia coli, Salmonella typhimurium, and five interspecies hybrids

The equilibrium and kinetic properties for the urea-induced unfolding of the alpha subunit of tryptophan synthase from Escherichia coli, Salmonella typhimurium, and five interspecies hybrids were compared to determine the role of protein folding in evolution. The parent proteins differ at 40 positions in the sequence of 268 amino acids, and the hybrids differ by up to 15 amino acids from the Escherichia coli alpha subunit. The results show that all the proteins follow the same folding mechanism and are consistent with a previously proposed hypothesis [Hollecker, M., & Creighton, T. E. (1983) J. Mol. Biol. 168, 409; Krebs, H., Schmid, F. X., & Jaenicke, R. (1983) J. Mol. Biol. 169, 619] that the folding mechanisms are conserved in homologous proteins. Analysis of the kinetic data suggests that the 15 positions at which the parent proteins differ in the amino folding unit, residues 1-188, do not play a role in a rate-limiting step in folding that has been previously identified as the association of the amino and carboxyl folding units [Beasty, A. M., Hurle, M. R., Manz, J. T., Stackhouse, T. S., Onuffer, J. J., & Matthews, C. R. (1986) Biochemistry 25, 2965]. One or more of the 25 positions at which the parent proteins differ in the carboxyl folding unit, residues 189-268, do appear to play a role in this same rate-limiting step.     

Effect of single amino acid replacements on the folding and stability of dihydrofolate reductase from Escherichia coli

The role of the secondary structure in the folding mechanism of dihydrofolate reductase from Escherichia coli was probed by studying the effects of amino acid replacements in two alpha helices and two strands of the central beta sheet on the folding and stability. The effects on stability could be qualitatively understood in terms of the X-ray structure for the wild-type protein by invoking electrostatic, hydrophobic, or hydrogen-bonding interactions. Kinetic studies focused on the two slow reactions that are thought to reflect the unfolding/refolding of two stable native conformers to/from their respective folding intermediates [Touchette, N. A., Perry, K. M., & Matthews, C. R. (1986) Biochemistry 25, 5445-5452]. Replacements at three different positions in helix alpha B selectively alter the relaxation time for unfolding while a single replacement in helix alpha C selectively alters the relaxation time for refolding. This behavior is characteristic of mutations that change the stability of the protein but do not affect the rate-limiting step. In striking contrast, replacements in strands beta F and beta G can affect both unfolding and refolding relaxation times. This behavior shows that these mutations alter the rate-limiting step in these native-to-intermediate folding reactions. It is proposed that the intermediates have an incorrectly formed beta sheet whose maturation to the structure found in the native conformation is one of the slow steps in folding.     

Effect of a single amino acid substitution on the folding of the alpha subunit of tryptophan synthase

The urea-induced unfolding of a missense mutant of the alpha subunit of tryptophan synthase from Escherichia coli involving the replacement of Gly by Glu at position 211 has been monitored by absorbance changes at 286 nm. Like the wild-type protein, the equilibrium unfolding curve demonstrates the presence of one or more stable intermediates. Comparison of these results with those from the wild-type alpha subunit [Matthews, C. R., & Crisanti, M. M. (1981) Biochemistry 20, 784] shows that the transition from the native conformation to the stable intermediates is displaced to higher urea concentration in the mutant alpha subunit; however, the transition from the intermediates to the unfolded form is unaffected. Kinetic studies show that the amino acid replacement slows the rate of unfolding by an order of magnitude. The effect on refolding rates is complex. One phase, previously assigned to proline isomerization [Crisanti, M. M., & Matthews, C. R. (1981) Biochemistry 20, 2700], is unaffected by the substitution. The rate of the second phase, which is urea dependent down to about 1 M urea, is slower than the corresponding phase in the wild-type protein by approximately a factor of 2. Below about 1 M urea, the rate of this phase becomes urea independent and identical with that of the wild-type alpha subunit. This change in urea dependence has been ascribed to a change in the nature of the rate-limiting step for this process from one involving folding to one involving proline isomerization. The results support the folding model for the alpha subunit proposed previously [Matthews, C. R., & Crisanti, M. M. (1981) Biochemistry 20, 784] and clarify the role of proline isomerization in limiting the rate of folding.     

Effects of the phenylalanine-22----leucine, glutamic acid-49----methionine, glycine-234----aspartic acid, and glycine-234----lysine mutations on the folding and stability of the alpha subunit of tryptophan synthase from Escherichia coli

The effects of four single amino acid replacements on the stability and folding of the alpha subunit of tryptophan synthase from Escherichia coli have been investigated by ultraviolet differences spectroscopy. In previous studies [Miles, E. W., Yutani, K., & Ogasahara, K. (1982) Biochemistry 21, 2586], it had been shown that the urea-induced unfolding at pH 7.8, 25 degrees C, proceeds by the initial unfolding of the less stable carboxyl domain (residues 189-268) followed by the unfolding of the more stable amino domain (residues 1-188). The effects of the Phe-22----Leu, Glu-49----Met, Gly-234----Asp, and Gly-234----Lys mutants on the equilibrium unfolding process can all be understood in terms of the domain unfolding model. With the exception of the Glu-49----Met replacement, the effects on stability are small. In contrast, the effects of three of the four mutations on the kinetics of interconversion of the native form and one of the stable partially folded intermediates are dramatic. The results for the Phe-22----Leu and Gly-234----Asp mutations indicate that these residues play a key role in the rate-limiting step. The Glu-49----Met mutation increases the stability of the native form with respect to that of the intermediate but does not affect the rate-limiting step. The Gly-234----Lys mutation does not affect either the stability or the kinetics of folding for the transition between native and intermediate forms. The changes in stability calculated from the unfolding and refolding rate constants agree quantitatively with those obtained from the equilibrium data. When considered with the results from a previous study on the Gly-211----Glu replacement [Matthews, C. R., Crisanti, M. M., Manz, J. T., & Gepner G. L. (1983) Biochemistry 22, 1445], it can be concluded that the rate-limiting step in the conversion of the intermediate to the native conformation involves either domain association or some other type of molecule-wide phenomenon.     

Proline isomerization and the slow folding reactions of the alpha subunit of tryptophan synthase from Escherichia coli

Previous studies on the refolding of the alpha subunit of tryptophan synthase from Escherichia coli assigned two slow refolding phases to rate-limiting isomerizations of two 'essential' proline residues, one in each of the two domains of the protein (Matthews, C.R., Crisanti, M.M., Manz, J.T. and Gepner, G.L. (1983) Biochemistry 22, 1445-1452). The double-jump experiment (Brandts, J.F., Halvorson, H.R. and Brennan, M. (1975) Biochemistry 14, 4953-4963) was used to further investigate this phenomenon. The reaction assigned to the carboxyl domain is consistent with the proline isomerization hypothesis. The amino domain process is more rapid than expected for proline isomerization and may reflect another type of slow folding reaction. The results permit a further refinement of the folding model for the alpha subunit and demonstrate the existence of a third unfolded species whose folding is not limited by either of these two reactions.     

Time-resolved fluorescence anisotropy study of the refolding reaction of the alpha-subunit of tryptophan synthase reveals nonmonotonic behavior of the rotational correlation time

Time-resolved fluorescence anisotropy of a bound extrinsic probe was studied in an effort to characterize dynamic properties of the transient partially folded forms that appear during the folding of the alpha-subunit of tryptophan synthase (alphaTS) from Escherichia coli. Previous studies have shown that alphaTS, a single structural domain, can be cleaved into autonomously folding amino- and carboxy-folding units comprising residues 1-188 and 189-268, respectively [Higgins, W., Fairwell, T., and Miles, E. W. (1979) Biochemistry 18, 4827-4835]. By use of a double-kinetic approach [Jones, B. E., Beechem, J. M., and Matthews, C. R. (1995) Biochemistry 34, 1867-1877], the rotational correlation time of 1-anilino-8-naphthalene sulfonate bound to nonpolar surfaces of folding intermediates was measured by time-correlated single photon counting at varying time delays following initiation of folding from the urea-denatured form by stopped-flow techniques. Comparison of the rotational correlation times for the full-length alphaTS and the amino-terminal fragment suggests that folding of the amino-terminal fragment and carboxy-terminal fragment is coordinated, not autonomous, on the milliseconds to seconds time scale. If a spherical shape is assumed, the apparent hydrodynamic radius of alphaTS after 5 ms is 26.8 A. The radius increases to 28.5 A by 1 s before decreasing to the radius for native alphaTS, 24.7 A, on a longer time scale (>25 s). Viewed within the context of the kinetic folding model of alphaTS [Bilsel, O., Zitzewitz, J. A., Bowers, K. E. , and Matthews, C. R. (1999) Biochemistry 38, 1018-1029], the initial collapse reflects the formation of an off-pathway burst-phase intermediate in which at least part of the carboxy folding unit interacts with the amino folding unit. The subsequent increase in rotational correlation time corresponds to the formation of an on-pathway intermediate that leads to the native conformation. The apparent increase in the radius for the on-pathway intermediate may reflect a change in the interaction of the two-folding units, thereby forming a direct precursor for the alpha/beta barrel structure.     

Role of diffusion in the folding of the alpha subunit of tryptophan synthase from Escherichia coli

The rate-limiting step in the folding of the alpha subunit of tryptophan synthase has been proposed to be the association of two folding units. To probe the role of diffusion in this rate-limiting step, the urea-induced unfolding and refolding of the protein was examined in the presence of a number of viscosity-enhancing agents. The analysis was simplified by studying the effect of these agents on folding unit dissociation, the rate-limiting unfolding reaction, and the reverse of the rate-limiting step in refolding. In the presence of ethylene glycol, the relaxation times for unfolding to the same final conditions increased with increasing concentration of the cosolvent. When the effects of the cosolvent on protein stability were taken into account, the rates were found to show a unitary linear dependence on the viscosity of the solution. Similar results were obtained with glycerol and low concentrations of glucose, demonstrating that the effect is general and not specific to any viscogenic agent. These results clearly demonstrate that the rate-limiting folding unit association/dissociation reaction in the alpha subunit of tryptophan synthase involves a diffusional process.     

Molecular dissection of the folding mechanism of the alpha subunit of tryptophan synthase: an amino-terminal autonomous folding unit controls several rate-limiting steps in the folding of a single domain protein.

The alpha subunit of tryptophan synthase (alphaTS) from Escherichia coli is a 268-residue 8-stranded beta/alpha barrel protein. Two autonomous folding units, comprising the first six strands (residues 1-188) and the last two strands (residues 189-268), have been previously identified in this single structural domain protein by tryptic digestion [Higgins, W., Fairwell, T., and Miles, E. W. (1979) Biochemistry 18, 4827-4835]. The larger, amino-terminal fragment, alphaTS(1-188), was overexpressed and independently purified, and its equilibrium and kinetic folding properties were studied by absorbance, fluorescence, and near- and far-UV circular dichroism spectroscopies. The native state of the fragment unfolds cooperatively in an apparent two-state transition with a stability of 3.98 +/- 0.19 kcal mol(-1) in the absence of denaturant and a corresponding m value of 1.07 +/- 0.05 kcal mol(-1) M(-1). Similar to the full-length protein, the unfolding of the fragment shows two kinetic phases which arise from the presence of two discrete native state populations. Additionally, the fragment exhibits a significant burst phase in unfolding, indicating that a fraction of the folded state ensemble under native conditions has properties similar to those of the equilibrium intermediate populated at 3 M urea in full-length alphaTS. Refolding of alphaTS(1-188) is also complex, exhibiting two detectable kinetic phases and a burst phase that is complete within 5 ms. The two slowest isomerization phases observed in the refolding of the full-length protein are absent in the fragment, suggesting that these phases reflect contributions from the carboxy-terminal segment. The folding mechanism of alphaTS(1-188) appears to be a simplified version of the mechanism for the full-length protein [Bilsel, O., Zitzewitz, J. A., Bowers, K.E, and Matthews, C. R.(1999) Biochemistry 38, 1018-1029]. Four parallel channels in the full-length protein are reduced to a pair of channels that most likely reflect a cis/trans proline isomerization reaction in the amino-terminal fragment. The off- and on-pathway intermediates that exist for both full-length alphaTS and alphaTS(1-188) may reflect the preponderance of local interactions in the beta/alpha barrel motif.     

Rate-determining folding and association reactions on the reconstitution pathway of porcine skeletal muscle lactic dehydrogenase after denaturation by guanidine hydrochloride

Reactivation of tetrameric porcine skeletal muscle lactic dehydrogenase after dissociation and extensive unfolding of the monomers by 6 M guanidine hydrochloride (Gdn . HCl) is characterized by sigmoidal kinetics, indicating a complex mechanism involving rate-limiting folding and association steps. For analysis of the association reactions, chemical cross-linking with glutaraldehyde may be used [Hermann, R., Jaenicke, R., & Rudolph, R. (1981) Biochemistry 20, 2195-2201]. The data clearly show that the formation of a dimeric intermediate is determined by a first-order folding reaction of the monomers with k1 = (8.0 +/- 0.1) x 10(-4) s-1. The rate constant of the association of dimers to tetramers which represents the second rate-limiting step on the pathway of reconstitution after guanidine denaturation, was then determined by reactivation and cross-linking experiments after dissociation in 0.1 M H3PO4 containing 1 M Na2SO4. The rate constant for the dimer association (which is the only rate-limiting step after acid dissociation) was k2 = (3.0 +/- 0.5) x 10(4) M-1 s-1. On the basis of the given two rate constants, the complete reassociation pattern of porcine lactic dehydrogenase after dissociation and denaturation in 6 M Gdn . HCl can be described by the kinetic model (formula: see text).     

Preformed secondary structure drives the association reaction of GCN4-p1, a model coiled-coil system

The structure of the transition state for the rate-limiting step in the folding and association of the homodimeric coiled-coil peptide GCN4-p1, was probed by mutational analysis. A series of quadruple amino acid replacements that spanned the helix propensity scale were made at the four external f positions in the heptad repeat. Equilibrium and kinetic circular dichroism studies demonstrate that both the stability and the unfolding and refolding rate constants vary with helix propensity but also reflect interactions of the altered side-chains with their local environments. Pairwise replacements and fragment studies show that the two C-terminal heptads are the likely source of the nucleating helices. Helix-helix recognition between preformed elements of secondary structure plays an important role in this fundamental folding reaction.     

Single amino acids in the lumenal loop domain influence the stability of the major light-harvesting chlorophyll a/b complex

The major light-harvesting complex of photosystem II (LHCIIb) is one of the most abundant integral membrane proteins. It greatly enhances the efficiency of photosynthesis in green plants by binding a large number of accessory pigments that absorb light energy and conduct it toward the photosynthetic reaction centers. Most of these pigments are associated with the three transmembrane and one amphiphilic alpha helices of the protein. Less is known about the significance of the loop domains connecting the alpha helices for pigment binding. Therefore, we randomly exchanged single amino acids in the lumenal loop domain of the bacterially expressed apoprotein Lhcb1 and then reconstituted the mutant protein with pigments in vitro. The resulting collection of mutated recombinant LHCIIb versions was screened by using a 96-well-format plate-based procedure described previously [Heinemann, B., and Paulsen, H. (1999) Biochemistry 38, 14088-14093], enabling us to test several thousand mutants for their ability to form stable pigment-protein complexes in vitro. At least one-third of the positions in the loop domain turned out to be sensitive targets; i.e., their exchange abolished formation of LHCIIb in vitro. This confirms our earlier notion that the LHCIIb loop domains contribute more specifically to complex formation and/or stabilization than by merely connecting the alpha helices. Among the target sites, glycines and hydrophilic amino acids are more prominently represented than hydrophobic ones. Specifically, the exchange of any of the three acidic amino acids in the lumenal loop abolishes reconstitution of stable pigment-protein complexes, suggesting that ionic interactions with other protein domains are important for correct protein folding or complex stabilization. One hydrophobic amino acid, tryptophan in position 97, has been hit repeatedly in independent mutation experiments. From the LHCIIb structure and previous mutational analyses, we propose a stabilizing interaction between this amino acid and F195 near the C-proximal end of the third transmembrane helix.     

Amino acid replacements can selectively affect the interaction energy of autonomous folding units in the alpha subunit of tryptophan synthase.

Amino acid replacements were made at the interface between two autonomous folding units in the alpha subunit of tryptophan synthase from Salmonella typhimurium to test their mutual interaction energy. The results of equilibrium studies of the urea-induced unfolding reaction of the wild-type and mutant proteins in which phenylalanine 22 is replaced by leucine, isoleucine, and valine can be understood in terms of a selective decrease in the interaction energy between the two folding units; the intrinsic stability of each folding unit is not significantly altered. Kinetic studies of the rate-limiting step in unfolding show that the interaction energy appears in the transition state preceding the native conformation. Comparisons of the individual effects of these nonpolar side chains show that both hydrophobic and steric effects play important roles in the interaction energy between the folding units. The implication of these results is that the high cooperativity observed in the folding of many globular proteins may be reduced by appropriate amino acid replacements.     

Tryptophan replacements in the trp aporepressor from Escherichia coli: probing the equilibrium and kinetic folding models.

Mutants of the dimeric Escherichia coli trp aporepressor are constructed by replacement of the two tryptophan residues in each subunit in order to assess the effects on equilibrium and kinetic fluorescence properties of the folding reaction. The three kinetic phases detected by intrinsic tryptophan fluorescence in refolding of the wild-type aporepressor are also observed in folding of both Trp 19 to Phe and Trp 99 to Phe single mutants, demonstrating that these phases correspond to global rather than local conformational changes. Comparison of equilibrium fluorescence (Royer, C.A., Mann, C.J., & Matthews, C.R., 1993, Protein Sci. 2, 1844-1852) and circular dichroism transition curves induced by urea shows that replacement of either Trp 19 or Trp 99 results in noncoincident behavior. Unlike the wild-type protein (Gittelman, M.S. & Matthews, C.R., 1990, Biochemistry 29, 7011-7020), tertiary and/or quaternary structures are disrupted at lower denaturant concentration than is secondary structure. The equilibrium results can be interpreted in terms of enhancement in the population of a monomeric folding intermediate in which the lone tryptophan residue is highly exposed to solvent, but in which substantial secondary structure is retained. The location of both mutations at the interface between the two subunits (Zhang, R.G., et al., 1987, Nature 327, 591-597) provides a simple explanation for this phenomenon.     

Catalysis of RNA cleavage by the Tetrahymena thermophila ribozyme. 2. Kinetic description of the reaction of an RNA substrate that forms a mismatch at the active site

The site-specific endonuclease reaction catalyzed by the ribozyme from the Tetrahymena pre-rRNA intervening sequence has been characterized with a substrate that forms a "matched" duplex with the 5' exon binding site of the ribozyme [G2CCCUCUA5 + G in equilibrium with G2CCCUCU + GA5 (G = guanosine); Herschlag, D., & Cech, T.R. (1990) Biochemistry (preceding paper in this issue)]. The rate-limiting step with saturating substrate is dissociation of the product G2CCCUCU. Here we show that the reaction of the substrate G2CCCGCUA5, which forms a "mismatched" duplex with the 5' exon binding site at position -3 from the cleavage site, has a value of kcat that is approximately 10(2)-fold greater than kcat for the matched substrate (50 degrees C, 10 mM MgCl2, pH 7). This is explained by the faster dissociation of the mismatched product, G2CCCGCU, than the matched product. With subsaturating oligonucleotide substrate and saturating G, the binding of the oligonucleotide substrate and the chemical step are each partially rate-limiting. The rate constant for the chemical step of the endonuclease reaction and the rate constant for the site-specific hydrolysis reaction, in which solvent replaces G, are each within approximately 2-fold with the matched and mismatched substrates, despite the approximately 10(3)-fold weaker binding of the mismatched substrate. This can be described as "uniform binding" of the base at position -3 in the ground state and transition state [Albery, W.J., & Knowles, J. R. (1976) Biochemistry 15, 5631-5640]. Thus, the matched substrate does not use its extra binding energy to preferentially stabilize the transition state.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of supercoiling and sequence context on operator DNA binding with lac repressor

The dissociation of the repressor-operator complex from a series of negatively supercoiled plasmid DNAs was examined as a function of the sequence context, orientation, and spacing. The plasmids were grouped into four classes, each with common sequence context. The highest dissociation rate constants were observed for the plasmids containing only a single operator (or pseudooperator) sequence, while approximately 10-fold lower rate constants were measured for plasmids with the I gene pseudooperator in conjunction with either the Z gene pseudooperator or the primary operator. Comparison of the behavior of these two classes of plasmids demonstrated the importance of two operator sequences and supported a model of DNA loop formation to stabilize the repressor-operator complex (Whitson, P. A., and Matthews, K. S. (1986) Biochemistry 25, 3845-3852; Whitson, P. A., Olson, J. S., and Matthews, K. S. (1986) Biochemistry 25, 3852-3858; Whitson, P. A., Hsieh, W. T., Wells, R. D., and Matthews, K. S. (1987) J. Biol. Chem. 262, 4943-4946; Kr?mer, H., Niem?ller, M., Amouyal, M., Revet, B., von Wilcken-Bergmann, B., and Müller-Hill, B. (1987) EMBO J. 6, 1481-1491). The third class, with intermediate dissociation rate constants, was comprised of plasmids which contained the primary operator and the higher affinity pseudooperator normally located in the Z gene. Neither the additional presence of the I gene pseudooperator nor the orientation of the primary operator relative to the Z gene pseudooperator significantly affected the dissociation rate constants. The binding characteristics of this group of plasmids demonstrated the essential role of the Z gene pseudooperator in the formation of intramolecular ternary complex and suggested an in vivo function for this pseudooperator. Plasmids containing two primary operator sequences were the class with lowest dissociation rate constants from lac repressor, and minimal effects of salt or spacing on dissociation of this class were observed. These data are consistent with formation of an intramolecular complex with a looped DNA segment stabilized by the combination of increased local concentration of binding sites and torsional stresses on the DNA which favor binding in supercoiled DNA.     

Molecular thermodynamic model to predict the alpha-helical secondary structure of polypeptide chains in solution.

The native state of a protein molecule in aqueous solutions represents one of the lowest states of Gibbs energy [Anfinsen, C.B. (1973) Science 181, 223-230]. Much progress has been made about the rules of protein folding [King, J. (1989) Chem. Eng. News 67, 32-54] and the dominant forces in protein folding [Dill, K.A. (1990) Biochemistry 29, 7133-7155]. However, the quantitative contributions of different Gibbs energy terms to protein stability remains a controversial issue [Moult, J., & Unger, R. (1991) Biochemistry 30, 3816-3824]. A molecular thermodynamic model has been proposed for the Gibbs energy of folding a residue in aqueous homopolypeptides from a random-coiled state to either the alpha-helix state or the beta-sheet state [Chen, C.-C., Zhu, Y., King, J.A., & Evans, L.B. (1992) Biopolymers 32, 1375-1392]. In this work, we present a generalization of the molecular thermodynamic model for the Gibbs energy of folding natural and synthetic heteropolypeptides from random-coiled conformations into alpha-helical conformations. The generalized model incorporates the intrinsic folding potential due to residue-solvent interactions, the cooperative folding effect due to residue-residue interactions, and the location and length of alpha-helices. The utility of the model was demonstrated by examining the stability of alpha-helical conformations of a number of natural polypeptides including C-peptide (residues 1-13) and S-peptide (residues 1-20) of RNase A (bovine pancreatic ribonuclease A), the P alpha fragment in BPTI (bovine pancreatic trypsin inhibitor), and synthetic polypeptides (the copolymers of different amino acid residues) including alanine-based peptides (16 or 17 residues long) in water. The computed Gibbs energies correspond well with the experimental data on helicity. The results also accounted for the effects of amino acid substitution and temperature on the stability of alpha-helical conformations of the test polypeptides.     

蛋白质折叠速率与其氨基酸序列极性的关系

以大肠杆菌60个蛋白酶以及几种常见病毒（SARS病毒、艾滋病病毒、丙型肝炎病毒及乙型肝炎病毒）各蛋白质序列中的所有α-螺旋和β-折叠片段为研究对象,计算了各片段的折叠速率和平均极性,分别在各物种的α-螺旋和β-折叠两类二级结构片段中分析了二者的相关性。得到结论：不论是大肠杆菌中的蛋白酶还是病毒蛋白,其中的两类氨基酸片段的平均极性与折叠速率都是极显著相关的：对于所有的α片段,二者呈线性正相关,而对于所有的β片段,二者成线性负相关。结果证实了在蛋白质折叠中,氨基酸的极性起着重要的作用。     

The tyrosine-225 to phenylalanine mutation of Escherichia coli aspartate aminotransferase results in an alkaline transition in the spectrophotometric and kinetic pKa values and reduced values of both kcat and Km

Tyrosine-225 is hydrogen-bonded to the 3'-hydroxyl group of pyridoxal 5'-phosphate in the active site of aspartate aminotransferase. Replacement of this residue with phenylalanine (Y225F) results in a shift in the acidic limb of the pKa of the kcat/KAsp vs pH profile from 7.1 (wild-type) to 8.4 (mutant). The change in the kinetic pKa is mirrored by a similar shift in the spectrophotometrically determined pKa of the protonated internal aldimine. Thus, a major role of tyrosine-225 is to provide a hydrogen bond that stabilizes the reactive unprotonated form of the internal aldimine in the neutral pH range. The Km value for L-aspartate and the dissociation constant for alpha-methyl-DL-aspartate are respectively 20- and 37-fold lower in the mutant than in the wild-type enzyme, while the dissociation constant for maleate is much less perturbed. These results are interpreted in terms of competition between the Tyr225 hydroxyl group and the substrate or quasi-substrate amino group for the coenzyme. The value of kcat in Y225F is 450-fold less than the corresponding rate constant in wild type. The increased affinity of the mutant enzyme for substrates, combined with the lack of discrimination against deuterium in the C alpha position of L-aspartate in Y225F-catalyzed transamination [Kirsch, J. F., Toney, M. D., & Goldberg, J. M. (1990) in Protein and Pharmaceutical Engineering (Craik, C. S., Fletterick, R., Matthews, C. R., & Wells, J., Eds.) pp 105-118, Wiley-Liss, New York], suggests that the rate-determining step in the mutant is hydrolysis of the ketimine intermediate rather than C alpha-H abstraction which is partially rate-determining in wild type.     

Structure-activity relationships in the oxidation of para-substituted benzylamine analogues by recombinant human liver monoamine oxidase A.

Monoamine oxidase A (MAO A) plays a central role in the oxidation of amine neurotransmitters. To investigate the structure and mechanism of this enzyme, recombinant human liver MAO A was expressed and purified from Saccharomyces cerevisiae. Anaerobic titrations of the enzyme require only 1 mol of substrate per mole of enzyme-bound flavin for complete reduction. This demonstrates that only one redox-active group (i.e., the covalent FAD cofactor) is involved in catalysis. The reaction rates and binding affinities of 17 para-substituted benzylamine analogues with purified MAO A were determined by steady state and stopped flow kinetic experiments. For each substrate analogue that was tested, the rates of steady state turnover (k(cat)) and anaerobic flavin reduction (k(red)) are similar in value. Deuterium kinetic isotope effects on k(cat), k(red), k(cat)/K(m), and k(red)/K(s) with alpha, alpha-[(2)H]benzylamines are similar for each substrate analogue that was tested and range in value from 6 to 13, indicating that alpha-C-H bond cleavage is rate-limiting in catalysis. Substrate analogue dissociation constants determined from reductive half-reaction experiments as well as from steady state kinetic isotope effect data [Klinman, J. P., and Matthews, R. G. (1985) J. Am. Chem. Soc. 107, 1058-1060] are in excellent agreement. Quantitative structure-activity relationship (QSAR) analysis of dissociation constants shows that the binding of para-substituted benzylamine analogues to MAO A is best correlated with the van der Waals volume of the substituent, with larger substituents binding most tightly. The rate of para-substituted benzylamine analogue oxidation and/or substrate analogue-dependent flavin reduction is best correlated with substituent electronic effects (sigma). Separation of the electronic substituent parameter (sigma) into field-inductive and resonance effects provides a more comprehensive treatment of the electronic correlations. The positive correlation of rate with sigma (rho approximately 2.0) suggests negative charge development at the benzyl carbon position occurs and supports proton abstraction as the mode of alpha-C-H bond cleavage. These results are discussed in terms of several mechanisms proposed for MAO catalysis and with previous structure-activity studies published with bovine liver MAO B [Walker, M. C., and Edmondson, D. E. (1994) Biochemistry 33, 7088-7098].     

A differential scanning calorimetric study of the thermal unfolding of mutant forms of phage T4 lysozyme.

In continuation of our earlier work on the effects of amino acid replacements on the thermodynamics of the thermal unfolding of T4 lysozyme [Kitamura, S., & Sturtevant, J. M. (1989) Biochemistry 28, 3788-3792; Connelly, P., Ghosaini, L., Hu, C.-Q., Kitamura, S., Tanaka, A., & Sturtevant, J. M. (1991) Biochemistry 30, 1887-1891; Hu, C.-Q., Kitamura, S., Tanaka, A., & Sturtevant, J. M. (1992) Biochemistry 31, 1643-1647], we report here a study by differential scanning calorimetry of the effects of five replacements at Ile3. Four of these replacements, those with Glu, Phe, Pro, and Thr, caused apparent destabilizations, while the replacement by Leu led to a small apparent stabilization. The largest observed destabilization (Ile3Pro) amounted to -3.0 kcal mol-1 in free energy at pH 2.00 and 38.8 degrees C (the denaturational temperature of the wild-type protein at this pH), and the largest stabilization amounted to +1.2 kcal mol-1 at pH 3.00 and 53.6 degrees C.