Formulation of a universal first-order rate constant for enzymatic reactions.

It is a common practice to employ k(cat)[E]0/K(m) as a first-order rate constant for the analysis of an enzymatic reaction, where [E]0 is the total enzyme concentration. I describe in this report a serious shortcoming in analyzing enzymatic reactions when kcat[E]0/K(m) is employed and show that k(cat)[E]0/K(m) can only be applied under very limited conditions. I consequently propose the use of a more universal first-order rate constant, k(cat)[ES](K)/[S]0, where [ES](K) is the initial equilibrium concentration of the ES-complex derived from [E]0, [S]0 and K(m). Employing k(cat)[ES](K)/[S]0 as the first-order rate constant enables all enzymatic reactions to be reasonably simulated under a wide range of conditions, and the catalytic and binding contributions to the rate constant of any enzyme can be determined under any and all conditions.

A protein's conformational stability is an immunologically dominant factor: evidence that free-energy barriers for protein unfolding limit the immunogenicity of foreign proteins.

Foreign protein Ags are incorporated into APCs and then degraded by endosomal proteases. The peptides are then mounted on MHC II molecules on the surfaces of APCs. The T cell-triggering response and, therefore, the immune response, were suggested to be governed by the degree of conformational stability of the foreign protein Ags. However, there is little evidence that a protein's conformational stability is an immunologically dominant factor. In this study, we show that a protein has a threshold of conformational stability to prevent the immunogenicity of foreign proteins. Inverse and linear correlations were found between the amount of IgG production against lysozymes and the free-energy change for the unfolding of lysozymes, based on the correlation between the free-energy changes of the protein unfolding and the amount of IgG production against lysozymes with different stabilities in mice using hen egg white lysozyme derivatives and mutant mouse lysozymes, in which the sequence between 107 and 116 is replaced with that of hen egg white lysozyme, which can produce autoantibodies in mice. Interestingly, the thresholds of free-energy changes for both lysozymes to prevent their immunogenicity were almost identical (21-23 kcal/mol). To confirm the results, we also showed that the cross-linking of Phl p 7, in which intact Phl p 7 has stability greater than ∼20 kcal/mol under physiological conditions, induced minimal IgG production in mice, whereas intact Phl p 7 was antigenic. From the above results, we suggest that protein conformational stability was an immunologically dominant factor.

Crystal structures of K33 mutant hen lysozymes with enhanced activities.

Using random mutagenesis, we previously obtained K33N mutant lysozyme that showed a large lytic halo on the plate coating Micrococcus luteus. In order to examine the effects of mutation of K33N on enzyme activity, we prepared K33N and K33A mutant lysozymes from yeast. It was found that the activities of both the mutant lysozymes were higher than those of the wild-type lysozyme based on the results of the activity measurements against M. luteus (lytic activity) and glycol chitin. Moreover, 3D structures of K33N and K33A mutant lysozyme were solved by X-ray crystallographic analyses. The side chain of K33 in the wild-type lysozyme hydrogen bonded with N37 involved in the substrate-binding region, and the orientation of the side chain of N37 in K33 mutant lysozymes were different in the wild-type lysozyme. These results suggest that the enhancement of activity in K33N mutant lysozyme was due to an alteration in the orientation of the side chain of N37. On the other hand, K33N lysozyme was less stable than the wild-type lysozyme. Lysozyme may sacrifice its enzyme activity to acquire the conformational stability at position 33.

Effect of buffer species on the unfolding and the aggregation of humanized IgG.

The aggregation propensity of humanized antibody after heat treatment is evaluated in the presence of six buffer species. The comparison under equivalent pH showed high aggregation propensity on phosphate and citrate buffer. In contrast, 2-(N-Morpholino) ethane sulfonate (MES), 3-(N-Morpholino) propane sulfonate (MOPS), acetate and imidazole buffer showed lower aggregation propensity than the above two buffers. Meanwhile, unfolding temperature evaluated by differential scanning calorimetry measurement was not altered among these buffer species. The light scattering analysis suggested that heat-denatured intermediate was aggregated slightly on MES and acetate buffer. Therefore, it was found that the different aggregation propensity among buffer species was caused from the aggregation propensity of heat-denatured intermediate rather than the unfolding temperature. Furthermore, it was revealed that the aggregation dependency on buffer species is accounted for by the specific molecular interaction between buffer and IgG, rather than the ionic strength. On the contrary, on the analyses of unfolding and aggregation propensity by molecular dissection of IgG into Fab and Fc fragments, aggregation propensity of Fc fragment on MES, acetate and phosphate buffer was almost the same as whole IgG. From the above results, it was suggested that the specific interaction between buffer molecule and Fc domain of IgG was involved in the aggregation propensity of heat-denatured IgG.

Amyloid formation in denatured single-mutant lysozymes where residual structures are modulated.

Reduced hen lysozyme has a residual structure involving long-range interaction. It has been demonstrated that a single mutation (A9G, W62G, W111G, or W123G) in the residual structure differently modulates the long-range interactions of reduced lysozyme. To examine whether such variations in the residual structure affect amyloid formation, reduced and alkylated mutant lysozymes were incubated under the amyloid-fibrillation condition. From the analyses of CD spectra and thioflavine T fluorescences, it was suggested that variation in residual structure led to different amyloid formation. Interestingly, the extent of amyloid formation did not always correlate with the extent to which the residual structure was maintained, resulting in the involvement of a hydrophobic cluster normally contained in W111 in the reduced lysozyme.

Effect of the structure of the denatured state of lysozyme on the aggregation reaction at the early stages of folding from the reduced form.

We previously demonstrated that the hydrophobic clusters present in hen lysozyme under denaturing conditions were disrupted by the mutation of Trp62 to Gly (W62G). In order to examine the effects of the structure of the denatured state of W62G lysozyme on folding, we analyzed the early events in the folding of reduced W62G lysozyme in detail. From the exchange measurements of disulfide bonds using the variants containing a pair of cysteine residues (1SS), it was found that the formation of disulfide bond in the W62G1SS lysozyme was not accompanied by a prominent interaction between amino acid residues, indicating that the disruption of the hydrophobic core led to the random folding at the early stages in the process of folding of the reduced lysozyme. On the other hand, analyses of the oxidative-renaturation of reduced W62G lysozymes, as well as measurements of the extent of aggregation of the reduced and carboxy amido methylated W62G lysozyme, indicated that the formation of an aggregate is more prominent in the reduced W62G lysozyme than in the reduced wild-type lysozyme. Moreover, a lag phase was detected in the oxidative-renaturation of reduced W62G lysozyme, as based on observations of the recovery of activity. The simulation of the folding process indicated that intermediates were present at the early stages in the folding of the reduced W62G lysozyme. These results suggest that the presence of the intermediates was derived from the random folding at the early stages in the folding process of reduced W62G lysozyme due to the disruption of the structure of the denatured state. Folding thus appears to have been kinetically delayed by these processes, which then led to the significant aggregation of reduced lysozyme. Moreover, from the analysis of amyloid aggregation of the reduced lysozymes, it was suggested that the disruption of the residual structure in denatured state by W62G mutation deterred the formation of the amyloid fibrils of lysozyme.

Analysis of internal motions of RNase T1 complexed with a productive substrate involving 15N NMR relaxation measurements.

The backbone dynamics of RNase T1 in the presence of exo-guanosine 2',3'-cyclophosphorothioate (exo-cGPS isomer), which is a productive substrate, and in the presence of 3'-guanylic acid (3'GMP), which is an nonproductive substrate, were examined using (15)N nuclear magnetic resonance. Although the X-ray crystal structure suggests that the modes of binding of these substrates to the active-site cleft are very similar, the order parameters in a number of regions in RNase T1 complexed with exo-cGPS isomer were different from those with 3'GMP. Moreover, the chemical exchange in line width observed for RNase T1 complexed with exo-cGPS isomer was also different from that observed for RNase T1 complexed with 3'GMP. From these results, we concluded that the internal motions in RNase T1 complexed with a productive substrate were not always identical to those in RNase T1 complexed with a nonproductive substrate.

Inputting information about amino acid residues in protein bioinformatics: a case study on predicting helical regions with a neural network.

The best way to introduce information about amino acid residues into calculations of protein bioinformatics was examined. That was done for predicting helical regions with a neural network. Several fundamental and instructive ways for information processing were developed and are described.

Derivation of a valid momentary first-order rate constant for kinetic and energetic analyses of enzymatic reactions.

To analyze enzymatic reactions energetically for comparison with non-enzymatic reactions (first order) under the same dimension, a method to derive valid momentary first-order rate constants for enzymatic reactions was developed. The momentary first-order rate constant, k enz0 = k cat[E'S']e,0/[S]0, was derived for an enzymatic reaction under a certain condition. It was shown that this rate constant is applicable for a wide range of enzymatic reactions. Utilizing this constant, one can conduct reliable kinetic and energetic analyses of enzymatic reactions.

Effect on catalysis by replacement of catalytic residue from hen egg white lysozyme to Venerupis philippinarum lysozyme.

Asn46Asp/Asp52Ser or Asn46Glu/Asp52Ser hen egg white lysozyme (HEL) mutant was designed by introducing the substituted catalytic residue Asp46 or Glu46, respectively, based on Venerupis philippinarum (Vp) lysozyme structure as a representative of invertebrate-type (i-type) lyzozyme. These mutations restored the bell-shaped pH-dependency of the enzyme activity from the sigmoidal pH-dependency observed for the Asp52Ser mutant. Furthermore both lysozyme mutants possessed retaining mechanisms like Vp lysozyme and HEL. The Asn46Glu/Asp52Ser mutant, which has a shorter distance between two catalytic residues, formed a glycosyl adduct in the reaction with the N-acetylglucosamine oligomer. Furthermore, we found the accelerated turnover through its glycosyl adduct formation and decomposition. The turnover rate estimated from the glycosyl formation and decomposition rates was only 20% of the observed hydrolysis rate of the substrate. Based on these results, we discussed the catalytic mechanism of lysozymes.

Protein unfolding at interfaces: slow dynamics of alpha-helix to beta-sheet transition.

A two-phase sequential dynamic change in the secondary structure of hen egg lysozyme (Lys) adsorbed on solid substrates was observed. The first phase involved fast conversion of alpha-helix to random/turns (within the first minute or at very low coverage or high substrate wettability) with no perceptible change in beta-sheet content. The second phase (1-1200 min), however, involved a relatively slow conversion from alpha-helix to beta-sheet without a noticeable change in random/turns. An important finding of this work is that the concentration of lysozyme in the adsorbed state has a substantial effect on the fractional content of secondary structures. Attenuated total reflection Fourier transform infrared (ATR/FTIR) spectroscopy, along with a newly-developed optimization algorithm for predicting the content of secondary structure motifs, was used to correlate the secondary structure and the amount of adsorbed lysozyme with the surface wettability of six different flat nanoporous substrates. Although three independent variables, surface wettability, solution concentration and time for adsorption, were used to follow the fractional structural changes of lysozyme, the results were all normalized onto a single plot with the amount adsorbed as the universal independent variable. Consequently, lateral interactions among proteins likely drive the transition process. Direct intermolecular force adhesion measurements between lysozyme and different functionalized self-assembled alkanethiol monolayers confirm that hydrophobic surfaces interact strongly with proteins. The lysozyme-unfolding pathway during early adsorption appears to be similar to that predicted by published molecular modeling results.

A method for the detection of asparagine deamidation and aspartate isomerization of proteins by MALDI/TOF-mass spectrometry using endoproteinase Asp-N.

A method was established for evaluating Asn deamidation and Asp isomerization/racemization. To detect the subtle changes in mass that accompany these chemical modifications, we used a combination of enzyme digestion by endoproteinase Asp-N, which selectively cleaves the N-terminus of L-alpha-Asp, and MALDI/TOF-mass spectrometry. To achieve better resolution, we employed digests of (15)N-labeled protein as an internal standard. To demonstrate the advantages of this method, we applied it to identify deamidated sites in mutant lysozymes in which the Asn residue is mutated to Asp. We also identified the deamidation or isomerization site of the lysozyme samples after incubating them under acidic or basic conditions.

Fluctuations in free or substrate-complexed lysozyme and a mutant of it detected on x-ray crystallography and comparison with those detected on NMR.

A mutant lysozyme in which Arg14 and His15 were deleted together exhibited higher activity toward glycol chitin than the wild-type lysozyme. Moreover, the mutant lysozyme, which is less stable than the wild-type lysozyme by 7 degrees C, showed a shift of temperature dependence of activity to the low temperature side compared with the wild-type lysozyme [Protein Eng. 7, 743-748 (1994)]. In the free enzyme, the internal motion of the mutant lysozyme was similar to that of the wild-type. The internal motions of the wild-type and mutant lysozymes in the enzyme-substrate complex increased more than those in the free enzymes. Moreover, the increased internal motions of the substrate-complexed mutant lysozyme were greater than those of the substrate-complexed wild-type lysozyme in several residues [J. Mol. Biol. 286, 1547-1565 (1999)]. The structure of the mutant lysozyme was very similar to that of the wild-type lysozyme. Both structures were also alike in the complex of the trimer of N-acetyl-D-glucosamine. The mobility from B-factors agreed to some degree with that from order parameters in the regions showing great mobility of the protein, but this was not the case in the regions showing fast motion. However, we came to the same conclusion that the increased activity of the mutant lysozyme is due to the increase in the fluctuation of the lysozyme molecule. B-factor and order parameter do not always exhibit harmony because the time-scale of the analysis of mobility is different. However, they are not incompatible but complementary for detecting precise protein motions.

Amino acid residues in subsites e and f responsible for the characteristic enzymatic activity of duck egg-white lysozyme.

We analyzed the enzymatic properties of duck egg-white lysozyme II (DEL), which differs from hen egg-white lysozyme (HEL) in nineteen amino acid substitutions. A substrate binding study showed that DEL binds to the substrate analog at subsites A-C in the same manner as HEL. However, the experimental time-courses of DEL against the substrate N-acetylglucosamine pentamer, (GlcNAc)(5), revealed remarkably enhanced production of (GlcNAc)(2) and reduced production of (GlcNAc)(1) as compared to in the case of HEL. Computer simulation of the DEL-catalyzed reaction suggested that the amino acid substitutions at subsites E and F (Phe34 to Tyr and Asn37 to Ser) caused the great alteration in the time-courses of DEL. Subsequently, the enzymatic reactions of mutants, in which Phe34 and Asn37 in HEL were converted to Tyr and Ser, respectively, were characterized. The time-courses of the F34Y mutant exhibited profiles similar to those of HEL. In contrast, the characteristics of the N37S mutant were different from those of HEL and rather similar to those of DEL; the order of the amounts of (GlcNAc)(1) and (GlcNAc)(2) was reversed in comparison with in the case of HEL. Enhanced production of (GlcNAc)(2) was also observed for the mutant protein, F34Y/N37S, with two substitutions. These results indicated that the substitution of Asn37 with Ser can account, at least in part, for the characteristic time-courses of DEL. Moreover, replacement of Asn37 with Ser reduced the rate constant of transglycosylation. The substitution of the Asn37 residue may affect the transglycosylation activity of HEL.

Amyloid-beta-protein (A beta) (25-35)-associated free radical generation is strongly influenced by the aggregational state of the peptides.

We investigated whether or not the Amyloid-beta-protein (A beta) itself spontaneously generates free radicals using electron spin resonance (ESR) spectroscopy while also monitoring the aggregational state of A beta and A beta-induced cytotoxicity. The present results demonstrated a four-line spectrum in the presence of A beta25-35 with N-tert-butyl-alpha-phenylnitrone (PBN) but not in the presence of PBN alone in phosphate-buffered saline (PBS). The fact that the four-line spectrum obtained for the A beta25-35/PBN in PBS was completely abolished in the presence of the iron-chelating agent Desferal demonstrated the observed four-line spectrum to be iron-dependent. On the other hand, A beta25-35 with PBN in phosphate buffer (PB) did not produce any definite four-line spectrum. the present results showed the amyloid fibril formation of A beta25-35 in PBS to be much higher than that of A beta25-35 in PB. Moreover, A beta-induced cytotoxicity assays showed A beta incubated in PBS to be more cytotoxic than that incubated in PB. These results thus demonstrate that A beta(25-35)-associated free radical generation is strongly influenced by the aggregational state of the peptides.

Effects of stereochemistry of sugars on protein stabilities.

We investigated thermal stabilities of four proteins in the presence of four kinds of sugars to analyze the mechanism of stabilization of proteins by additives. These proteins were stabilized by the addition of sugars, and the degree of stabilization correlated to the partial molar isentropic compressibility of the sugar.

[Enjoying research on lysozymes for 42 years].

I have pursued research on lysozymes for 42 years. During that time, I made Several new findings, some of them by chance. My enjoyment of the following areas is reviewed: the story of tryptophan; protease digestion mechanisms; peptide mapping with RP-HPLC; gene engineering; renaturation of protein; catalytic residues; fluctuation and function; stabilization; folding; antigenecity; tolerance; and various lysozymes.

[Foundation of the bases for protein research and its application to the pharmaceutical science field].

This paper reviews the results of basic research conducted by the author's group to determine appropriate methods to develop protein-based drugs. These include production strategies, elucidation of physiologic function, improving existing pharmaceuticals, de novo design, and protein reconstruction. The antigenicity of modified proteins and methods to induce antigenic protein tolerance are also described.

Effect of the conformational stability of the CH2 domain on the aggregation and peptide cleavage of a humanized IgG.

To examine the effect of the conformational stability of the CH2 domain on aggregation and peptide cleavage of a humanized IgG1, we carried out size exclusion chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis analyses of incubated sample solutions. By comparing the residual percentage of monomer after incubation at 60 and 80°C at various pH levels, we found that aggregation and peptide cleavage of the humanized IgG1 occurred during long incubation at 60°C under acidic conditions. Next, we confirmed cleavage of the Asp272-Pro273 peptide bond in the CH2 domain. Comparison of the cleavage rates of the IgG1 monomer and a peptide containing the same Asp-Pro sequence revealed that the conformational stability of the CH2 domain retards cleavage of the Asp272-Pro273 peptide bond at 60°C and pH 4.0. The finding of aggregation and peptide cleavage of the humanized IgG1 after long incubation at 60°C under acidic conditions was supported by another finding: there were lower unfolding temperatures of the CH2 domain at pH 4.0 and 5.0. We conclude that the conformational stability of the CH2 domain is closely related to aggregation and peptide cleavage of the humanized IgG1 under acidic conditions. We also found that the 2-[N-morpholino] ethane sulfonate buffer inhibits aggregation of the IgG1 at pH 4.0-5.0 and 7.0-8.0.

Refolding of an unstable lysozyme by gradient removal of a solubilizer and gradient addition of a stabilizer.

Earlier, we formally established an effective refolding procedure for a protein by gradient removal of a solubilizer such as urea [Maeda et al. (1995) Effective renaturation of reduced lysozyme by gentle removal of urea. Protein Eng. 8, 201-205]. However, this procedure was less effective for unstable proteins. We developed here an excellent method to add protein stabilizer so as to get reasonable amounts of folded protein under the concentration of solubilizer where the unstable protein does not form aggregate. We examined many stabilizers and found that 60% of a concentrated (2.5 mg/ml) unstable protein can be refolded using 40% glycerol as the best stabilizer. This procedure can be widely applicable for the refolding of unstable proteins.