Biomedical subjects
Publications and source records attributed to J K Hwang.
We report a case of Wells' syndrome (eosinophilic cellulitis) with acute cutaneous swelling followed by indolent infiltration. The histopathology is characterized by a dense infiltrate of eosinophils and "flame figures" in the dermis. The electron microscopic findings are peculiar. This case responded well to oral antihistamines. The skin signs and symptoms disappeared completely two weeks later.
Recent advances in genetic engineering have led to a growing acceptance of the fact that enzymes work like other catalysts by reducing the activation barriers of the corresponding reactions. However, the key question about the action of enzymes is not related to the fact that they stabilize transition states but to the question to how they accomplish this task. This work considers the catalytic reaction of serine proteases and demonstrates how one can use a combination of calculations and experimental information to elucidate the key contributions to the catalytic free energy. Recent reports about genetic modifications of the buried aspartic group in serine proteases, which established the large effect of this group (but could not determine its origin), are analyzed. Two independent methods indicate that the buried aspartic group in serine proteases stabilizes the transition state by electrostatic interactions rather than by alternative mechanisms. Simple free energy considerations are used to eliminate the double proton-transfer mechanism (which is depicted in many textbooks as the key catalytic factor in serine proteases). The electrostatic stabilization of the oxyanion side of the transition state is also considered. It is argued that serine proteases and other enzymes work by providing electrostatic complementarity to the changes in charge distribution occurring during the reactions they catalyze.
Genetic engineering is a powerful tool for exploring correlations between structure and function in proteins, but as yet we are unable to use it for effective protein design. One of the most interesting examples, which would seem to be obvious, is reversing the polarity of an ion pair. Changing a positively charged protein group, that provides a strong binding for negative substrates, to a negative group is expected to provide an effective binding site for a positively charged substrate. But several recent experiments on aspartate aminotransferase, trypsin and aspartate transcarbamoylase (Schachman, H. K. personal communication) have indicated that polarity reversal is not so successful. Here we argue that the same factors that make the enzyme an effective system for the (-+) pair will make it a much less effective system for the (+-) pair. We also point out that the unusually low effective dielectric constant (epsilon approximately equal to 13) for the (-+) interaction is due to its microenvironment and this will destabilize a (+-) arrangement having an entirely different dielectric constant (epsilon approximately equal to 80). The calculations presented here evaluate the energetics of ion pairs in protein active sites on a semiquantitative level. This is particularly important when dealing with strong, functionally important interactions that are difficult to evaluate with macroscopic models.
A combination of the empirical valence bond method and a free energy perturbation approach is used to simulate the activity of genetically modified enzymes. The simulations reproduce in a semiquantitative way the observed effects of mutations on the activity and binding free energies of trypsin and subtilisin. This suggests that we are approaching a stage of quantitative structure-function correlation of enzymes. The analysis of the calculations points towards the electrostatic energy of the reacting system as the key factor in enzyme catalysis. The changes in the charges of the reacting system and the corresponding changes in "solvation" free energy (generalized here as the interaction between the charges and the given microenvironment) are emphasized. It is argued that a reliable evaluation of these changes might be sufficient for correlating structure and catalysis. The use of free energy perturbation methods and thermodynamic cycles for evaluation of solvation energies and reactivity is discussed, pointing out our early contributions. The apparent elaborated nature of our treatment is clarified, explaining that such a treatment is essential for consistent calculations of chemical reactions in polar environments. The problems associated with seemingly more rigorous quantum mechanical methods are discussed, emphasizing the inconsistency associated with using gas phase charge distributions. The importance of dynamic aspects is examined by evaluating the autocorrelation of the protein "reaction field" on the reacting substrate. It is found that, at least in the present case, dynamic effects are not important. The nature of the catalytic free energy is considered, arguing that the protein provides preoriented dipoles (polarized to stabilize the transition state charge distribution) and small reorganization energy, thus reducing the activation free energy. The corresponding catalytic free energy is related to the folding free energy, which is being invested in aligning the active site dipoles.
The catalytic free energy and binding free energies of the native and the Asn-155----Thr, Asn-155----Leu, and Asn-155----Ala mutants of subtilisin are calculated by the empirical valence bond method and a free energy perturbation method. Two simple procedures are used; one "mutates" the substrate, and the other "mutates" the enzyme. The calculated changes in free energies (delta delta G not equal to cat and delta delta Gbind) between the mutant and native enzymes are within 1 kcal/mol of the corresponding observed values. This indicates that we are approaching a quantitative structure-function correlation. The calculated changes in catalytic free energies are almost entirely due to the electrostatic interaction between the enzyme-water system and the charges of the reacting system. This supports the idea that the electrostatic free energy associated with the changes of charges of the reacting system is the key factor in enzyme catalysis.
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We report eight additional cases of focal fibrocartilaginous dysplasia (FFCD) in the proximal tibia (five), distal ulna (one), and distal femur (two). Spontaneous, complete resolution of the lesion was observed in two tibiae and one ulna. Three tibial lesions with genu varum deformity were managed with osteotomy. Two femoral FFCDs caused persistent or progressive deformity: one genu valgum with patellar dislocation, and one genu varum. These patients underwent concomitant deformity correction and lengthening by the Ilizarov method. The final results were satisfactory in all patients except one, who underwent valgus tibial osteotomy and developed mild postoperative genu valgum. The analysis of a total of 46 cases in the literature and our experience suggests that (a) FFCD has a wide histopathologic spectrum, ranging from purely dense, fibrous tendon-like tissue to benign fibrocartilaginous tissue; (b) at least 45% of tibial FFCD demonstrates progressive, spontaneous resolution; (c) in contrast, femoral and humeral FFCDs appear to have a slim possibility of spontaneous regression of the deformity; and (d) corrective osteotomy is indicated when the deformity is increasing or persistent, or when the existing deformity is severe enough to jeopardize adjacent joint mechanics and alignment.