摘要:

AbstractInterfacial catalysis is a necessary consequence for all enzymes that act on amphipathic substrates with a strong tendency to form aggregates in aqueous dispersions. In such cases the catalytic event occurs at the interface of the aggregated substrate, the overall turnover at the interface is processive, and it is influenced the molecular organization and dynamics of the interface. Such enzymes can access the substrate only at the interface because the concentration of solitary monomers of the substrate in the aqueous phase is very low. Moreover, the microinterface between the bound enzyme and the organized substrate not only facilitates formation of the enzyme–substrate complex, but a longer residence time of the enzyme at the substrate interface also promotes high catalytic processivity.Binding of the enzyme to the substrate interface as an additional step in the overall catalytic turnover permits adaptation of the Michaelis–Menten formalism as a basis to account for the kinetics of interfacial catalysis. As shown for the action of phospholipase A2on bilayer vesicles, binding equilibrium has two extreme kinetic consequences. During catalysis in the scooting mode the enzyme does not leave the surface of the vesicle to which it is bound. On the other hand, in the hopping mode the absorption and desorption steps are a part of the catalytic turnover.In this minireview we elaborate on the factors that control binding of pig pancreatic phospholipase A2to the bilayer interface. Binding of PLA2 to the interface occurs through ionic interactions and is further promoted by hydrophobic interactions which probably occur along a face of the enzyme, with a hydrophobic collar and a ring of cationic residues, through which the catalytic site is accessible to substrate molecules in the bilayer. An enzyme molecule binds to the surface occupied by about 35 lipid molecules with an apparent dissociation constant of less than 0.1 pM for the enzyme on anionic vesicles compared to 10 mM on zwitterionic vesicles. Results at hand also show that aggregation or acylation of the protein is not required for the high affinity binding or catalytic interaction at the interf

ISSN:0887-3585    

DOI:10.1002/prot.340090402

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY

摘要:

AbstractHemocyanins are copper‐containing proteins that transport oxygen in a variety of invertebrates. Considerable evidence has accumulated that arthropodan hemocyanins are multimers of a fundamental hexameric unit. X‐Ray crystallographic structure determination has revealed that the hemocyanin molecule from the spiny lobsterPanulirus interruptusis a single hexamer having 32 point group symmetry. Using crystals of subunit II, one of 8 polypeptide types comprising the octahexameric hemocyanin of the horseshoe crabLimulus polyphemus, and the molecular replacement method for crystallographic phase determination we show that subunit II forms assemblies with the same hexameric quaternary structure as the wholePanulirushemocyanin molecule. Observation of the same hexameric motif in two widely separated species provides strong additional evidence that this quaternary structural unit is a universal building block of arthropodan hemocyan

ISSN:0887-3585    

DOI:10.1002/prot.340090403

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY

摘要:

AbstractChemical shifts of resonances of specific protons in the1H NMR spectrum of thermally denatured hen lysozyme have been determined by exchange correlation with assigned native state resonances in 2D NOESY spectra obtained under conditions where the two states are interconverting. There are subtle but widespread deviations of the measured shifts from the values which would be anticipated for a random coil; in the case of side chain protons these are virtually all net upfield shifts and it is shown that this may be the averaged effect of interactions with aromatic rings in a partially collapsed denatured state. In a very few cases, notably that of two sequential tryptophan residues, it is possible to interpret these effects in terms of specific, local interresidue interactions. Generally, however, there is no correlation with either native state shift perturbations or with sequence proximity to aromatic groups. Diminution of most of the residual shift perturbations on reduction of the disulfide cross‐links confirms that they are not simply effects of residues adjacent in the sequence. Similar effects of chemical denaturants, with the disulfides intact, demonstrate that the shift perturbations reflect an enhanced tendency to side chain clustering in the thermally denatured state. The temperature dependences of the shift perturbations suggest that this clustering is noncooperative and is driven by small, favorable enthalpy changes. While the extent of conformational averaging is clearly much greater than that observed for a homologous protein, α‐lactalbumin, in its partially folded “molten globule” state, the results clearly show that thermally denatured lysozyme differs substantially from a random coil, principally in that it is partially hydrophobically c

ISSN:0887-3585    

DOI:10.1002/prot.340090404

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY

摘要:

AbstractThe active site of dienelactone hydrolase (DLH), a microbial enzyme of the β‐ketoadipate pathway, has been conclusively located using a combination of crystallographic, biochemical, and genetic techniques. DLH hydrolyzes a dienelactone to maleylacetate and has esterase activity onp‐nitrophenyl acetate and trans‐cinnamoyl imidazole. The identification of Cys‐123 as containing the essential thiol confirms the localization of the active site as suggested by the crystal structure of DLH, and disproves an earlier hypothesis regarding its location. Two mutant proteins have been engineered in which Cys‐123 has been converted to a serine (C123S DLH) and an alanine (C123A DLH), respectively. C123S DLH (Km= 9900±2300 μM;Vmax= 4.4±0.8 μmol/min‐mg) displays burst kinetics withp‐nitrophenyl acetate and is 10% as active as DLH (Km= 170±7 μM;Vmax= 21.1±0.4 μmol/min‐mg). C123A DLH is inactive. The structures of DLH, C123S DLH, and C123A DLH have been refined at 1.8, 2.2, and 2.0 Å, respectively. Comparison of the structures of these proteins demonstrates that the only differences between them are centered at residue 123. The structures of the active sites of DLH, papain, and subtilisin are similar and are suggestive of the three enzymes having evolved convergently to similar active sites with s

ISSN:0887-3585    

DOI:10.1002/prot.340090405

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY

摘要:

AbstractThe folding of a polypeptide into a parallel (α/β)8barrel (which is also called a circularly permuted β8α8barrel) has been investigated in terms of energy minimization. According to the arrangement of hydrogen bonds between two neighboring β‐strands of the central barrel therein, such an α/β barrel structure can be folded into six different types: (1) left‐tilted, left‐handed crossover; (2) left‐tilted, right‐handed crossover; (3) nontilted, left‐handed crossover; (4) nontilted, right‐handed crossover; (5) right‐tilted, left‐handed crossover; and (6) right‐tilted, right‐handed crossover. Here “tilt” refers to the orientational relation of the β‐strands to the axis of the central β‐barrel, and “crossover” to the βαβ folding connection feature of the parallel β‐barrel. It has been found that the right‐tilted, right‐handed crossover α/β barrel possesses much lower energy than the other five types of α/β barrels, elucidating why the observed α/β barrels in proteins always assume the form of right tilt and right‐handed crossover connection. As observed, the β‐strands in the energy‐minimized right‐tilted, right‐handed crossover (α/β)8‐barrel are of strong right‐handed twist. The value of root‐mean‐square fits also indicates that the central barrel contained in the lowest energy (α/β)8structure thus found coincides very well with the observed 8‐stranded parallel β‐barrel in triose phosphate isomerase (TIM). Furthermore, an energetic analysis has been made demonstrating why the right‐tilt, right‐handed crossover barrel is the most stable structure. Our calculations and analysis support the principle that it is possible to account for the main features of frequently occurring folding patterns in proteins by means of con

ISSN:0887-3585    

DOI:10.1002/prot.340090406

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY

ISSN:0887-3585    

DOI:10.1002/prot.340090401

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY