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Journal Issue - Volume 9 Issue 1 (2000)

Abstract Substrate‐assisted catalysis (SAC) is the process by which a functional group in a substrate contributes to catalysis by an enzyme. SAC has been demonstrated for representatives of three major enzyme classes: serine proteases, GTPases, and type II restriction endonucleases, as well as lysozyme and hexose‐1‐phosphate uridylyltransferase. Moreover, structure‐based predictions of SAC have been made for many additional enzymes....

Abstract Whereas previously we have successfully utilized the folding funnels concept to rationalize binding mechanisms (Ma B, Kumar S, Tsai CJ, Nussinov R, 1999, Protein Eng 12:713–720) and to describe binding (Tsai CJ, Kumar S, Ma B, Nussinov R, 1999, Protein Sci 8:1181–1190), here we further extend the concept of folding funnels, illustrating its utility in explaining enzyme pathways, multimolecular associations, and allostery. This...

Abstract Inspection of high resolution three‐dimensional (3D) structures from the protein database reveals an increasing number of cis‐Xaa‐Pro and cis‐Xaa‐Yaa peptide bonds. However, we are still far from being able to predict whether these bonds will remain cis upon single‐site substitution of Pro or Yaa and/or cleavage of a peptide bond close to it in the sequence. We have chosen oxidized Escherichia coli thioredoxin (Trx), a...

Abstract The crystal structures of four active site‐directed thrombin inhibitors, 1–4, in a complex with human α‐thrombin have been determined and refined at up to 2.0 Å resolution using X‐ray crystallography. These compounds belong to a structurally novel family of inhibitors based on a 2,3‐disubstituted benzo[b]thiophene structure. Compared to traditional active‐site directed inhibitors, the X‐ray crystal structures of these complexes ...

Abstract The dihydrolipoamide succinyltransferase (E2o) component of the α‐ketoglutarate dehydrogenase complex catalyzes the transfer of a succinyl group from the S‐succinyldihydrolipoyl moiety to coenzyme A. E2o is normally a 24‐mer, but is found as a trimer when E2o is expressed with a C‐terminal [His]6 tag. The crystal structure of the trimeric form of the catalytic domain (CD) of the Escherichia coli E2o has been solved to 3.0 Å resolution...

Abstract The X‐ray crystallographic structures of two mutants (K206Q and H207E) of the N‐lobe of human transferrin (hTF/2N) have been determined to high resolution (1.8 and 2.0 Å, respectively). Both mutant proteins bind iron with greater affinity than native hTF/2N. The structures of the K206Q and H207E mutants show interactions (both H‐bonding and electrostatic) that stabilize the interaction of Lys296 in the closed conformation,...

Abstract As an alternative method to study the heterotropic mechanism of Escherichia coli aspartate transcarbamoylase, a series of nucleotide analogs were used. These nucleotide analogs have the advantage over site‐specific mutagenesis experiments in that interactions between the backbone of the protein and the nucleotide could be evaluated in terms of their importance for function. The ATP analogs purine 5′‐triphosphate (PTP), 6‐chloropurine...

Abstract The conformational changes during the photocycle of the photoactive yellow protein have been the subject of many recent studies. Spectroscopic measurements have shown that the photocycle also occurs in a crystalline environment, and this has been the basis for subsequent Laue diffraction and cryocrystallographic studies. These studies have shown that conformational changes during the photocycle are limited to the...

Abstract Parvalbumins constitute a class of calcium‐binding proteins characterized by the presence of several helix‐loop‐helix (EF‐hand) motifs. In a previous study (Revett SP, King G, Shabanowitz J, Hunt DF, Hartman KL, Laue TM, Nelson DJ, 1997, Protein Sci 7:2397–2408), we presented the sequence of the major parvalbumin isoform from the silver hake (Merluccius bilinearis) and presented spectroscopic and structural information on the excised “EF‐hand”...

Abstract Turkey ovomucoid third domain (OMTKY3) is a canonical inhibitor of serine proteinases. Upon complex formation, the inhibitors fully exposed P1 residue becomes fully buried in the preformed cavity of the enzyme. All 20 P1 variants of OMTKY3 have been obtained by recombinant DNA technology and their equilibrium association constants have been measured with six serine proteinases. To rationalize the trends observed in this data set, high...

Abstract Molecular dynamics calculations provide a method by which the dynamic properties of molecules can be explored over timescales and at a level of detail that cannot be obtained experimentally from NMR or X‐ray analyses. Recent work (Philippopoulos M, Mandel AM, Palmer AG III, Lim C, 1997, Proteins 28:481–493) has indicated that the accuracy of these simulations is high, as measured by the correspondence of parameters extracted ...

Abstract A divalent metal ion, such as Mn2+, is required for the catalytic reaction and allosteric regulation of pig heart NAD‐dependent isocitrate dehydrogenase. The enzyme is irreversibly inactivated and cleaved by Fe2+ in the presence of O2 and ascorbate at pH 7.0. Mn2+ prevents both inactivation and cleavage. Nucleotide ligands, such as NAD, NADPH, and ADP, neither prevent nor promote inactivation or cleavage of the enzyme by Fe2+. The...

Abstract An analysis of the folding of the 94 residue tenth fibronectin type III (fnIII) domain of human fibronectin (FNfn10) is presented. Use of guanidine isothiocyanate as a denaturant allows us to obtain equilibrium and kinetic data across a broad range of denaturant concentrations that are unavailable in guanidine hydrochloride. Equilibrium unfolding experiments show that FNfn10 is significantly more stable than has been...

Abstract The nitrogenase enzyme of Klebsiella pneumoniae consists of two separable proteins, each with multiple subunits and one or more oxygen sensitive metallocenters. The wild‐type nitrogenase proteins are stable to electrophoresis in high concentrations of urea under anaerobic conditions. Addition of Mg+2 and ADP greatly increases the stability of the smaller Fe protein (from 6 M for full unfolding), an effect directly analogous...

Abstract The rate of macromolecular surface formation in yeast iso‐2 cytochrome c and its site‐specific mutant, N52I iso‐2, has been studied using a monoclonal antibody that recognizes a tertiary epitope including K58 and H39. The results indicate that epitope refolding occurs after fast folding but prior to slow folding, in contrast to horse cytochrome c where surface formation occurs early. The antibody‐detected (ad) kinetic phase...

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