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Biomedical subjects

A Bulychev

Publications and source records attributed to A Bulychev.

12 recordsLinked to original sources
PubMed2000

A stabilized molten globule protein.

A predominant conformational isomer of non-native alpha-lactalbumin (alpha-LA) has been purified by thermal denaturation of the native alpha-LA using the technique of disulfide scrambling. This unique isomer retains a substantial content of alpha-helical structure. It is stabilized by two native disulfide bonds within the alpha-helical domain and two scrambled non-native disulfide bonds at the beta-sheet domain. This denatured isomer of alpha-LA exhibits structural characteristics that are consistent with the well-documented molten globule state. The ability to prepare a stabilized and structurally defined molten globule provides a useful model for studying the folding and unfolding pathways of proteins.

Circular Dichroism↗
PubMed2000

The underlying mechanism for the diversity of disulfide folding pathways.

The disulfide folding pathway of bovine pancreatic trypsin inhibitor (BPTI) is characterized by the predominance of folding intermediates with native-like structures. Our laboratory has recently analyzed the folding pathway(s) of four 3-disulfide-containing proteins, including hirudin, potato carboxypeptidase inhibitor, epidermal growth factor, and tick anticoagulant peptide. Their folding mechanism(s) differ from that of BPTI by 1) a higher degree of heterogeneity of 1- and 2-disulfide intermediates and 2) the presence of 3-disulfide scrambled isomers as folding intermediates. To search for the underlying causes of these diversities, we conducted kinetic analyses of the reductive unfolding of these five proteins. The experiment of reductive unfolding was designed to evaluate the relative stability and interdependence of disulfide bonds in the native protein. It is demonstrated here that among these five proteins, there exists a striking correlation between the mechanism(s) of reductive unfolding and that of oxidative folding. Those proteins with their native disulfide bonds reduced in a collective and simultaneous manner exhibit both a high degree of heterogeneity of folding intermediates and the accumulation of scrambled isomers along the folding pathway. A sequential reduction of the native disulfide bonds is associated with the presence of predominant intermediates with native- like structures.

Aprotinin↗
PubMed1999

Inhibition of the broad spectrum nonmetallocarbapenamase of class A (NMC-A) beta-lactamase from Enterobacter cloacae by monocyclic beta-lactams.

beta-Lactamases hydrolyze beta-lactam antibiotics, a reaction that destroys their antibacterial activity. These enzymes, of which four classes are known, are the primary cause of resistance to beta-lactam antibiotics. The class A beta-lactamases form the largest group. A novel class A beta-lactamase, named the nonmetallocarbapenamase of class A (NMC-A) beta-lactamase, has been discovered recently that has a broad substrate profile that included carbapenem antibiotics. This is a serious development, since carbapenems have been relatively immune to the action of these resistance enzymes. Inhibitors for this enzyme are sought. We describe herein that a type of monobactam molecule of our design inactivates the NMC-A beta-lactamase rapidly, efficiently, and irreversibly. The mechanism of inactivation was investigated by solving the x-ray structure of the inhibited NMC-A enzyme to 1.95 A resolution. The structure shed light on the nature of the fragmentation of the inhibitor on enzyme acylation and indicated that there are two acyl-enzyme species that account for enzyme inhibition. Each of these inhibited enzyme species is trapped in a distinct local energy minimum that does not predispose the inhibitor species for deacylation, accounting for the irreversible mode of enzyme inhibition. Molecular dynamics simulations provided evidence in favor of a dynamic motion for the acyl-enzyme species, which samples a considerable conformational space prior to the entrapment of the two stable acyl-enzyme species in the local energy minima. A discussion of the likelihood of such dynamic motion for turnover of substrates during the normal catalytic processes of the enzyme is presented.

Anti-Bacterial Agents↗
PubMed1999

X-ray structure of the Asn276Asp variant of the Escherichia coli TEM-1 beta-lactamase: direct observation of electrostatic modulation in resistance to inactivation by clavulanic acid.

The clinical use of beta-lactam antibiotics combined with beta-lactamase inactivators, such as clavulanate, has resulted in selection of beta-lactamases that are insensitive to inactivation by these molecules. Therefore, therapeutic combinations of an enzyme inactivator and a penicillin are harmless for bacteria harboring such an enzyme. The TEM beta-lactamase variants are the most frequently encountered enzymes of this type, and presently, 20 variants are designated as inhibitor-resistant TEM ("IRT") enzymes. Three mutations appear to account for the phenotype of the majority of IRT enzymes, one of them being the Asn276Asp substitution. In this study, we have characterized the kinetic properties of the inhibition process of the wild-type TEM-1 beta-lactamase and of its Asn276Asp variant with the three clinically used inactivators, clavulanic acid (clavulanate), sulbactam, and tazobactam, and we report the X-ray structure for the mutant variant at 2.3 A resolution. The changes in kinetic parameters for the interactions of the inhibitors with the wild-type and the mutant enzymes were more pronounced for clavulanate, and relatively inconsequential for sulbactam and tazobactam. The structure of the Asn276Asp mutant enzyme revealed a significant movement of Asp276 and the formation of a salt bridge of its side chain with the guanidinium group of Arg244, the counterion of the inhibitor carboxylate. A water molecule critical for the inactivation chemistry by clavulanate, which is observed in the wild-type enzyme structure, is not present in the crystal structure of the mutant variant. Such structural changes favor the turnover process over the inactivation chemistry for clavulanate, with profound phenotypic consequences. The report herein represents the best studied example of inhibitor-resistant beta-lactamases.

Asparagine↗
PubMed1999

Unfolding of hirudin characterized by the composition of denatured scrambled isomers.

The native core structure of hirudin, a thrombin specific inhibitor, contains 24 hydrogen bonds, two stretches of beta-sheet and three disulfide bonds. Hirudin unfolds in the presence of denaturant and thiol catalyst by shuffling its native disulfide bonds and converting to scrambled structures that consist of 11 identified isomers. The composition of scrambled isomers, which characterizes the structure of denatured hirudin, varies as a function of denaturing conditions. The unfolding pathway of hirudin has been constructed by quantitative analysis of scrambled isomers unfolded under increasing concentrations of various denaturants. The results demonstrate a progressive expansion of the polypeptide chain and the existence of a structurally defined stable intermediate along the pathway of unfolding.

Amino Acid Sequence↗
PubMed1999

Class C beta-lactamases operate at the diffusion limit for turnover of their preferred cephalosporin substrates.

It has been suggested that class C beta-lactamases have evolved to carry out a metabolic reaction other than hydrolysis of beta-lactam antibiotics. It is demonstrated in the present study that the class C beta-lactamase from Enterobacter cloacae P99 has reached the diffusion limit in its ability to hydrolyze its preferred cephalosporin substrates. The increase in the solution viscosity by addition of a microviscogen (sucrose) caused the decline in the parameter kcat/Km for hydrolysis of cephaloridine and cephalosporin C (approximately 2.5-fold at a relative viscosity of 2.9). A similar increase in viscosity has no effect on the turnover rate of the poorer substrates cefepime and penicillin G. Addition of a macroviscogen (polyethylene glycol) to the reaction mixture did not change the rate of turnover for any of the substrates tested because in this case the viscogen would not interfere with the motion of small molecules, as was expected. Therefore, it would appear that the driving force behind the evolution of this class C beta-lactamase and, in principle, other enzymes of this class is indeed the functional reaction of this enzyme as a drug resistance factor.

Cephalosporins↗
PubMed1998

Templates for design of inhibitors for serine proteases: application of the program DOCK to the discovery of novel inhibitors for thrombin.

The program DOCK was used to search for novel inhibitors for alpha-thrombin. Four among the top twelve best scoring compounds from the Cambridge Structural Data Base inhibited this enzyme, and three of them inhibited alpha-thrombin in a competitive mode. These molecules are expected to serve as general templates for structural elaboration in targeting diverse serine proteases for selective inhibition.

Binding Sites↗
PubMed1998

Effects of some biologically active compounds on phagosome-lysosome fusion in peritoneal macrophages of mice.

Effects of biologically active compounds bilirubin (BR), farmorubicin (FR), and chelerythrine (CR) on phagosome-lysome (P-L) fusion in mouse peritoneal macrophages were studied using fluorescent dye acridine orange as lysosomal labelling and yeast cells as target. It was found that all three compounds tested enhanced P-L fusion. To investigate mechanisms of these effects, changes in fluidity of rat liver lysosomal membranes under influence of BR, FR and CR were studied by measuring fluorescence intensity, lifetime, and polarization of DPH or TMA-DPH incorporated in isolated rat liver lysosomes. In order to characterize the cytoskeleton changes under the action of these biologically active compounds F-actin content in peritoneal macrophages of mice was determined. Our results demonstrate that BR action induces a decrease in DPH and TMA-DPH polarization, FR increases DPH and TMA-DPH polarization, and CR causes only an increase in TMA-DPH polarization in lysosomal membranes. All three compounds tested increase F-actin content in peritoneal macrophages. Thus, the effect of BR on P-L fusion is connected with increasing fluidity of lysosomal membranes and the cytoskeleton changes. The enhancement of P-L fusion under the action of FR and CR can most likely be explained by changes of the cytoskeleton state.

Actins↗
PubMed1994

Precursor-mediated opening of translocation pores in chloroplast envelopes.

Direct electrical measurements on native chloroplast envelopes reveal that a full-length chloroplast precursor protein causes an increase in the conductivity of the envelope membranes, due to its transit sequence. The conductivity is not influenced by a truncated precursor protein incapable of efficient translocation, suggesting precursor-mediated opening of translocation pores in chloroplast envelopes.

Chloroplasts↗