On the basis of the equations derived previously [Tsou, C. L. (1965) Sheng Wu Hua Hsueh Yu Sheng Wu Wu Li Hsueh Pao 5, 398-408, 409-417] for the substrate reaction during the course of enzyme modification, the kinetic behavior of the system chymotrypsin-substrate-modifier has been studied. The kinetics of benzoyltyrosine ester hydrolysis during the course of irreversible inhibition of the enzyme has been found to be in satisfactory agreement with equations obtained previously. The apparent rate constant between the enzyme and an irreversible inhibitor can be easily obtained in one single experiment by following the course of substrate hydrolysis in the presence of the inhibitor. The results are also in accord with the assumption that diisopropyl fluorophosphate can be classified as an irreversible competitive inhibitor. For both phenylmethanesulfonyl fluoride and L-1-[(p-toluene-sulfonyl)amino]-2-phenylethyl chloromethyl ketone, the inhibition has been found to be in agreement with the kinetics of the complexing type; i.e., a noncovalent enzyme-inhibitor complex is formed before irreversible enzyme modification. Both the equilibrium constants for the complex formation and the first-order rate constants for the irreversible modification step have been determined also by following the course of substrate hydrolysis in the presence of the irreversible inhibitor.
Camellia, comprising more than 200 species, is the type genus of the family Theaceae. Currently, the interspecies relationship of the economically important genus is still a matter of great debate and controversy. In an attempt to help settle this dispute using molecular phylogeny, we analyzed ITS sequences of 112 species of Camellia. The maximum parsimony and Bayesian trees grouped these species into eight major clades and four isolates. The current study supported the monophyly of sections Thea and Furfuracea, a merged section of Theopsis and Eriandra and the formation of section Oleifera by H, -t. Chang (Flora of Reipulicae Popularis Sinicae. Tomus 49 (3), Science Press, China). The study suggested the polyphyletic nature of the sections Camellia, Paracamellia, Pseudocamellia, and Tuberculata and the paraphyletic nature of the section Chrysantha but did not support the sectional status of the three small sections, Archecamellia, Piquetia, and Sterocarpus. We also discuss the results in terms of morphology, geographic distribution and the results from an earlier molecular phylogeny analysis.
ments are aligned in all possible ways. For each alignment, a score is obtained by counting +1 for a match, -pi for a mismatch, and -6 for a letter inserted or deleted (a gap).For example, AGCACT and AGGT can be aligned as AGCACT AG-G-T to receive score S = 3 -gialso be aligned as 26. They can AGGACT AGGT-to receive score S = 2 -2g -25. The score Mmn(x,y), which is defined to be the maximum of all the scores obtained for all possible pairs of segments I and J, is then calculated by an algorithm whose computing time is proportional to the product of m and n.To calculate the probability of those large values of Mmn(x,y) for which the similarity is significant, one has to know, at least approximately, the distribution of Mmn(X,y) under the assumption that x and y are unrelated. That is, the letters xl,..., Xm, Yt'* ,y,, are independently chosen with the same distribution from the alphabet {A,C,G,T}. Karlin and Altschul (7) obtained approximations for the probabilities of large values of Mmn(XY) for the case 5 = oo (that is, without gaps), assuming the expected score of two letters to be negative. Arratia, Gordon, and Waterman (8) considered the score Mmn(t), which is the maximum of the scores obtained by considering only those pairs of segments I and J of a given length t, for the case g = 0. They established approximations for Mmn(t) under certain mild conditions by the method of Poisson approximation.The set of all values of the parameters (p,6) can be divided into two regions, S, and S2, such that for m = n, the growth of Mnn(x,y) is proportional to n in S, and the growth of Mn(x,y) is proportional to log n in S2. The cases considered by Karlin and Altschul (7) and Arratia, Gordon, and Waterman (8) are in the logarithmic region. The work of the latter (8) has provided a basis for Waterman and Vingron (9) to use the Poisson clumping heuristic of Aldous (10) to calculate the probabilities of large values of Mmn(xY) in the entire logarithmic region. Let us see how the method of Poisson approximation is applied in the problem of Arratia, Gordon, and Waterman (8). Because p = 0, the score for each pair of the segments I and J of a given length t is just the number of matches. Let s be a given positive integer. Associate an event with 380 each pair of I and J. If the score is at least s for a particular pair of I and J, we say that the associated event occurs. The number of events that occur, say, U is the number of those scores which are at least s. Therefore, P[Mmn(t) < S] = P(U = 0). We would have been done if the distribution of U was approximately Poisson with mean, say, X*. For then, we would have had P[Mmn(t) . s] = 1 -P(U = O) = 1 -e A .However, this is not the case. The events associated with the I's and the J's occur in clumps. By the Poisson clumping heuristic of Aldous (10), it is the number of clumps that is expected to have approximately the Poisson distribution. So we declump and modify the events so as to obtain events which are associated with the clumps. Let W denote the number of clumps that occur. T...
Equilibrium and kinetic studies of the guanidine hydrochloride induced unfolding-refolding of dimeric cytoplasmic creatine kinase have been monitored by intrinsic fluorescence, far ultraviolet circular dichroism, and I-anilinonaphthalene-8-sulfonate binding. The GuHCl induced equilibrium-unfolding curve shows two transitions, indicating the presence of at least one stable equilibrium intermediate in GuHCl solutions of moderate concentrations. This intermediate is an inactive monomer with all of the thiol groups exposed. The thermodynamic parameters obtained by analysis using a three-state model indicate that this intermediate is similar in energy to the fully unfolded state. There is a burst phase in the refolding kinetics due to formation of an intermediate within the dead time of mixing (1 5 ms) in the stopped-flow apparatus. Further refolding to the native state after the burst phase follows biphasic kinetics. The properties of the burst phase and equilibrium intermediates were studied and compared. The results indicate that these intermediates are similar in some respects, but different in others. Both are characterized by pronounced secondary structure, compact globularity, exposed hydrophobic surface area, and the absence of rigid side-chain packing, resembling the "molten globule" state. However, the burst phase intermediate shows more secondary structure, more exposed hydrophobic surface area, and more flexible side-chain packing than the equilibrium intermediate. Following the burst phase, there is a fast phase corresponding to folding of the monomer to a compact conformation. This is followed by rapid assembly to form the dimer. Neither of the equilibrium unfolding transitions are protein concentration dependent. The refolding kinetics are also not concentration dependent. This suggests that association of the subunits is not rate limiting for refolding, and that under equilibrium conditions, dissociation occurs in the region between the two unfolding transitions. Based upon the above results, schemes of unfolding and refolding of creatine kinase are proposed.Keywords: creatine kinase; dimerization of subunits; folding intermediate; kinetics; molten globule; protein foldingIn spite of the accumulation of a large number of experimental studies, protein folding remains one of the most challenging subjects in structural biology. Characterization of folding intermediates is considered an important strategy for the elucidation of the mechanism of protein folding. A common equilibrium intermediate, the "molten globule" (MG) state, has been detected between the native (N) and the fully unfolded (U) states for many proteins (Fink, 1995; Ptitsyn, 1995). The MG state is characterized by pronounced secondary structure, compact globularity, exposed hydrophobic surface, and the absence of rigid side-chain packing
Protein disulfide isomerase (PDI) catalyzes the formation of native disulfides of peptide chains from either the reduced form or randomly joined disulfides. So that thiols situated at distant parts of the polypeptide chain can be joined together to form the native disulfides, the polypeptide chain has to be folded, at least to some extent, into the native conformation. It is suggested that PDI promotes folding of the chains as well as formation of the disulfides and plays a role similar to the chaperones in the folding process. PDI is known to be a multifunctional protein and capable of nonspecific peptide binding. These properties are closely connected to its possible function as a chaperone. Thioredoxin, which has an active site sequence similar to that of PDI but lacks the property of peptide binding, is much less efficient as a disulfide isomerase.
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