On the basis of the alternatives of direct inter-enzyme transfer vs. dissociation followed by random diffusion, two kinetic models for metabolite transfer between consecutive enzymes are developed. These two models are readily distinguishable experimentally for the transfer of 1,3-diphosphoglycerate (1,3-P2G) between glyceraldehyde-3-phosphate dehydrogenase (GPDH) and 3-phosphoglycerate kinase (PGK). Since 1,3-P2G is exceedingly tightly bound to PGK, the kinetics of its transfer to GPDH are predictably different for each of these two models. Our experiments unambiguously demonstrate that 1,3-P2G is directly transferred between these two enzymes via an enzyme-substrate-enzyme complex. This direct transfer is described by a Michaelis-Menten scheme in which PGK . 1,3-P2G is the "substrate" for GPDH. At high concentrations of PGK . 1,3-P2G, the transfer reaction becomes nearly PGK . 1,3-P2G concentration independent. The rate of the transfer reaction is activated 3.5-fold by saturating quantities of ATP and 20-fold by saturating quantities of 3-PG. Evidence is presented that the PGK . 1,3-P2G complex is structurally distinct from either PGK itself or other PGK . ligand complexes.

The imidazole of chromophoric p-(dimethylamino)benzoic acid, DABIm, reacts with the serine protease alpha-chymotrypsin in the pH range of 4-7 to form a stable acyl intermediate that gives very good resonance-enhanced Raman spectra. The resonance Raman and absorption spectra of the acyl enzyme intermediate have been compared with the spectra of simple model compounds such as the corresponding chromophoric methyl ester, aldehyde, and imidazole. The resonant Raman and ultraviolet absorption spectra of these simple chromophoric model compounds change considerably with the solvent. However, each of the model compounds exhibits a linear correlation between the maximum wavelength of absorption and the frequency of the carbonyl vibration. The observed values of the acyl intermediate do not fall on the line for the methyl ester but rather on the line for the aldehyde. This shows that the chromophoric serine ester of the acyl enzyme behaves differently than an ordinary ester, which cannot be explained as a solvent effect. Thermal unfolding of the acyl enzyme brings the spectroscopic parameters close to those of the model ester. We conclude that it is the specific conformation of the native enzyme and not solvent effects that change the spectroscopic properties of the acyl chromophore. It is reasonable that these changes arise from the same forces that cause the catalytic events. The carbonyl frequencies of a series of para-substituted benzoyl methyl esters show a remarkably linear correlation with the rate of deacylation of the corresponding acyl enzymes.

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The reaction of hen egg white lysozyme with chitotriose and chitohexose has been investigated at temperatures to below -100 degrees C by using aqueous methanol and dimethyl sulfoxide cryosolvents. Initial investigations involving the effects of increasing cosolvent concentration on the catalytic and structural properties of the enzyme indicated that both methanol and dimethyl sulfoxide cryosolvents, at subzero temperatures, had no adverse effects on lysozyme. Time-dependent changes in the fluorescence emission of the enzyme, under nonturnover conditions, permitted the detection of two intermediates in the reaction with the trimer and three intermediates in the case of the hexamer. The fastest reaction observed for both substrates was complete within minutes at -90 degrees C and is attributed to initial substrate binding, followed by rapid isomerization to form a "loose" ES complex. The subsequent, slower reactions correspond to successive isomerizations of ES to "tighter" complexes. From dye binding displacement reactions, and comparison of dissociation constants, it is concluded that the predominant mode of binding for the hexamer at subzero temperature is productive. Rate and dissociated constants were determined for the observed reactions as a function of temperature, pH, and cosolvent concentration. Overall the reaction pathway in methanol cryosolvents at subzero temperatures appears to be very similar to that determined from investigations in aqueous solution at ambient temperatures using a variety of rapid reaction techniques. Conditions necessary to accumulate and stabilize each of the observed intermediates have been determined, thus permitting further studies to delve more deeply into the nature of the intermediates.