A continuation of research aimed at describing the catalytic mechanism of the phosphoglucomutase reaction at the atomic level is posed. X-ray diffraction studies of the crystalline enzyme and complexes thereof will provide primary data. Supporting data will be obtained from solution studies of the following: the interaction between the enzymic phosphate group and the active site metal ion; bonding making/breaking in the transition state; the importance of selected amino acid side chains that line the catalytic cleft. One of the first goals will be to improve the current molecular model of the enzyme by collecting and processing higher-resolution diffraction data. A recently identified procedure that produces an improved lattice during elimination of the salt used in crystal growth will be developed further and employed. Use of this procedure should allow formation of various complexes of the crystalline enzyme that could not be obtained otherwise because of the salt present in untreated crystals. (The physical basis for the apparent annealing that accompanies this treatment will be probed, as a side issue -- and possibly its generality, along with procedures for improving the crystal growth process.) A variety of substrate, substrate analog, and transition state-analog complexes, also involving alternative metal ions, will be studied in the crystal phase by utilizing electron density- difference maps. The objective will be to identify the basis of the >1010-fold substrate binding-induced rate effect and the >1010-fold Mg2+- induced activation previously observed. An evaluation of bonding within the vanadate group of the transition-state analog complex via Raman spectroscopy and the use of EPR spectroscopy to assess binding-induced changes in interaction between bound Mn2+ and the phosphate group of the enzyme, after labelling it with 17O, and NMR spectrometry with the 113Cd enzyme will aid in interpreting X-ray diffraction results. An assessment of the 18O-kinetic isotope effect in a reaction where transfer of the (- PO32-) group of the enzyme to a suitable acceptor is rate-limiting also will facilitate interpretation of models obtained from X-ray diffraction studies, including that of a bound transition-state analog. The identity of several surface residues within the unusually large active site cleft of phosphoglucomutase will be altered by site-directed mutagenesis, with initial emphasis on residues close to the catalytic site that seem too far away to be involved directly in catalysis. Kinetically interesting mutant proteins will be subjected to a thorough physical/chemical evaluation.