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The heme moiety of AHb1 and AHb2 is characterized to gain insight into structure-function relationships. Both AHb1 and AHb2 reveal different interactions of the distal cavity residues with both the heme and ligands.

The time course of CO rebinding to human hemoglobin also exhibits a biphasic geminate rebinding when the protein is entrapped in wet nanoporous silica gel. Potential hydrophobic docking cavities within the alpha and beta chains are identified and the connection between the two xenon sites in the hydrophobic pocket is relatively open, explaining the only slight change in the energetics of ligand migration between the two sites due to decreased protein mobility with viscosity.

A nanosecond pH-jump technique coupled with simultaneous transient absorption and fluorescence emission detection was used to characterize the dynamics of the acid-induced spectral changes in the GEPmut2 chromophore. Results indicate that the faster transient is associated with proton binding from the solution, while the second process, smaller in amplitude, is attributed to structural rearrangement of the amino acids surrounding the chromophore.

Research has been conducted on the human hemoglobins. The preparation of these hemoglobins with the substitution at a single residue has been carried out and their reaction kinetics with the carbon monoxide has been measured.d

A study was conducted to characterize the functions of Asp60 of the alpha-subunit in allosteric communication between the tryptophan synthase alpha and beta-subunits. An imaging plate system was utilized to determine diffraction data at 95 K. Results indicated that alpha-Asp60 supported a critical function in the closure of the loop 6 and in allosteric communication between the alpha and beta-subunits.

An investigation of pyridoxal 5'-phosphate (PLP)-O-acetylserine sulfhydrylase (OASS) intermediates was conducted to probe the active site microenvironment and conformational events at different stages of catalysis through the excitation in the spectral range where aromatic amino acids absorb. Upon excitation at either 420 or 330 nm, the external aldimines with L-cysteine and L-serine exhibit a structured emission centered at 490 nm with a shoulder at 530 nm.

Research shows that a highly distorted cofactor can be bound by D-amino acid oxidase (DAAO), helping the enzyme to act as a highly efficient oxidase. DAAO also has a flexible substrate binding site. It would appear from the imino tryptophan binding mode that direct hydride transfer to the flavin N5 atom from C-alpha causes substrate oxidation. Catalysis is probably assisted by loop closure as the hydrophobicity of the active site is enhanced to improve the hydride transfer reaction. A crystallographic analysis is presented.

The equilibrium between the two main catalytic species in a reaction between L-serine and the Salmonella typhimurium tryptophan synthase alpha-2-beta-2-complex is regulated by an ionization of the two groups. The relative concentrations of the two species are dependent on pH and alpha-subunit ligands. The formation of the alpha-aminoacrylate Schiff base from external aldimine, a catalytic species, requires heat. There is a relation between the conversion of external aldimine and proton binding. The protons resemble alpha-subunit ligands in their allosteric effect.

Binding of monovalent cations produces conformational changes affecting the functional properties of tryptophan synthase alpha2beta2 complex from Salmonella typhimurium, and the functional changes are retained in the crystalline state. In the presence of cations such as lithium, sodium or ammonium, the phosphorescence decay of the tryptophan-177 beta residue situated near the active site on the beta region between the alpha and beta subunits is biphasic and there is an increase in the lifetime of the enzyme. The role of the cations appears to be structural rather than catalytic. They may act to stabilize conformations of enzymes and intermediates.

Polarized absorption microspectrophotometry shows that the presence of chloride decreases the affinity for oxygen of human hemoglobin Rothschild in its T quaternary structure, while Hb Rothschild has a higher affinity for oxygen in the absence of chloride. Chloride does not affect the oxygen affinity of human hemoglobin A (HbA). The Hb crystals studied were grown from polyethylene glycol solutions which had low salt concentrations. Chloride binding to Hb Rothschild lowers oxygen affinity in both the alpha and beta chains.

Polarized optical absorption measurements were odtained using a microspectrophotometer to characterize reversible oxygen binding for single crystals of hemoglobin in the T quarternary structure. In particular, the degree of cooperativity within the quarternary structure, the inequivalence in oxygen binding to the alpha and beta hemes and the effect of pH on the crystal binding curves were examined. The prediction of a noncooperative crystal binding curve that is pH-dependent for the maintenance of the T-quarternary structure is tested.

A study was conducted on regulatory signal communication and conformational changes in alpha- and beta- subunits of the tryptophan synthase alpha2beta2 complex from Salmonella typhimurium. Results showed that the binding of DL-alpha-glycerol 3-phosphate influences the structure of the alpha-subunit and promotes conformational change leading to structural rigidity in the beta-subunit in the presence of coenzyme. The mechanisms and implications of allosteric regulation between alpha- and beta- subunits are also presented.

A study was conducted to characterize tryptophan and coenzyme fluorescence and phosphorescence present in tryptophan synthase from Salmonella typhimurium. The coenzyme was found to be well-protected against thermal quenching and strong intensity from the ketoenamine tautomer band. The position 177 tryptophan was found to be highly luminiscent and possessed a narrow and well-defined O-O vibrational band. Trp-177 phospherescence was also observed to be long-lived even when exposed to ambient temperature.

A detailed study was conducted to characterize mutant enzyme O-Acetylserine sulfhydrylase which is a pyridoxal 5'-phosphate (PLP)-dependent enzyme and the residues ESB, T68, S69, N71, and Q142 were changed to alanine to probe the contribution these residues towards the active site closing and orientation of PLP. The obtained data suggested that interaction of intermediates with the substrate-binding loop comprised of T68, S69, G70, and N71 and Q142 gives a properly aligned Michaelis complex and facilitates the [beta]-elimination reaction.

The control of the development of various ionizable residues in the catalytic mechanism of tryptophan synthase [alpha.sub.2][beta.sub.2] complexes obtained from Salmonella typhimurium is explained.

Kinetics and spectral studies are presented to characterize the B-isozyme in order to explain the functional properties of B-isozyme of O-acetylserine sulfhydrylase (OASS-B) and also to understand the differences in structure-function relationships between OASS-A and OASS-B. The results have suggested a higher degree of conformational flexibility linked to catalysis for the O-acetylserine sulfhydrylase B-isozyme.

The Raman UV resonance spectroscopies are used to study the conformational properties of both the deoxy and deoxy-turned-carbonmonoxy (CO) derivatives of Hb-A derived from encapsulation protocols. The results show that the initial deoxy-HbA populations are conformationally indistinguishable with respect to the encapsulation protocol.

Results point out that the catalysis of the reversible hydrolytic cleavage of L-tryptophan by the tryptophan indole-lyase from Proteus vulgaris is mediated by amino acid residues aspartate-133 and histidine-458. Data indicate that the residues participate in the elimination reaction of L-tryptophan with aspartate-133 forming a hydrogen bond with the indole NH of the substrate and histidine-458 hydrogen bonded to aspartate-133.

Research has been conducted on human adult hemoglobin. The authors report that they have probed functionally distinct conformations of this hemoglobin via the use of diliganded and deoxy derivatives of human adult hemoglobin?s Fe-Zn symmetric hybrids, and that the progression of conformational and functional properties is consistent with the picture in which the T quaternary structure can accommodate tertiary conformations.

Computational titration results are presented of the titration analysis of a multiprotic HIV-1 protease-ligand complex. This calculation method can be applied to any protein-ligand complexes with structural data.