Immobilized active monomers of D-glyceraldehyde-3-phosphate dehydrogenase from rabbit skeletal muscles and their coenzyme-binding properties

Experimental conditions favouring the dissociation of tetrameric rabbit muscle D-glyceraldehyde-3-phosphate dehydrogenase into active monomers were elaborated. The urea-induced dissociation of the tetramer was shown to be a stepwise process (in 2 M urea only dimers are formed; an increase in urea concentration up to 3 M causes the splitting of the dimers into monomers). The specific activity of immobilized monomers in the glyceraldehyde-3-phosphate oxidation reaction does not differ from that of the parent immobilized tetrameric form. The tetrameric enzyme molecule binds the coenzyme with a negative cooperativity (the first two NAD+ molecules bind with KD below 0.1 microM; for the third and fourth molecules the dissociation constant was determined to be equal to 5.5 +/- 1.5 microM (50 mM medinal buffer, 10 mM sodium phosphate, pH 8.2). The cooperativity of NAD+ binding is preserved in the immobilized preparation of tetrameric dehydrogenase. The immobilized monomers bind NAD+ with KD of 1.6 +/- 1.0 microM. The experimental results are consistent with the hypothesis according to which the association of catalytically active subunits into a tetramer changes their coenzyme-binding properties in such a way that the first two NAD+ molecules bind more firmly to a tetramer than to a monomer, whereas the third and the fourth NAD+ molecules bind less firmly.     

Yeast glyceraldehyde-3-phosphate dehydrogenase. Evidence that subunit cooperativity in catalysis can be controlled by the formation of a complex with phosphoglycerate kinase

Sepharose-bound tetrameric, dimeric and monomeric forms of yeast glyceraldehyde-3-phosphate dehydrogenase were prepared, as well as immobilized hybrid species containing (by selective oxidation of an active center cysteine residue with H2O2) one inactivated subunit per tetramer or dimer. The catalytic properties of these enzyme forms were compared in the forward reaction (glyceraldehyde-3-phosphate oxidation) and reverse reaction (1,3-bisphosphoglycerate reductive dephosphorylation) under steady-state conditions. In the reaction of glyceraldehyde-3-phosphate oxidation, immobilized monomeric and tetrameric forms exhibited similar specific activities. The hybrid-modified dimer contributed on half of the total activity of a native dimer. The tetramer containing one modified subunit possessed 75% of the activity of an unmodified tetramer. In the reaction of 1,3-bisphosphoglycerate reductive dephosphorylation, the specific activity of the monomeric enzyme species was nearly twice as high as that of the tetramer, suggesting that only one-half of the active centers of the oligomer were acting simultaneously. Subunit cooperativity in catalysis persisted in an isolated dimeric species. The specific activity of a monomer associated with a peroxide-inactivated monomer in a dimer was equal to that of an isolated monomeric species and twice as high as that of a native immobilized dimer. The specific activity of subunits associated with a peroxide-inactivated subunit in a tetramer did not differ from that of a native immobilized tetramer; this indicates that interdimeric interactions are involved in catalytic subunit cooperativity. A complex was formed between the immobilized glyceraldehyde-3-phosphate dehydrogenase and soluble phosphoglycerate kinase. Three monomers of phosphoglycerate kinase were bound per tetramer of the dehydrogenase and one per dimer. Evidence is presented that if the reductive dephosphorylation of 1,3-bisphosphoglycerate proceeds in the phosphoglycerate kinase - glyceraldehyde-3-phosphate dehydrogenase complex, all active sites of the latter enzyme act independently, i.e. subunit cooperativity is abolished.     

Cooperative properties of D-glyceraldehyde-3-phosphate dehydrogenase]

The structure of the active center of glyceraldehyde-3-phosphate dehydrogenase and the arrangement of subunits in the tetrameric molecule is delineated. The mechanism of cooperative effects in the oligomer is considered, and the involvement of various regions of the active center and of different-subunit contact area in the realization of the cooperative phenomena is discussed. A special attention is paid to the effect of NAD+ bound to one of the subunits of the tetramer on the structure of an adjacent subunit and to the problem of the participation of the coenzyme in the creation of anion-binding sites of the enzyme. The conditions of reversible dissociation of the tetrameric apoenzyme molecule into dimers are depicted, and the role of NAD+ in the organization of the quaternary structure of the dehydrogenase is discussed. The problem of catalytic activity of the dimeric form of the enzyme is argued.     

Factors affecting coenzyme binding and subunit interactions in glyceraldehyde-3-phosphate dehydrogenase.

There is no evidence, at pH 9.4, of negative cooperativity in the binding of NAD+ or NADH to rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phorphorylating), EC 1.2.1.12) nor in the binding of acetyl pyridine adenine dinucleotide at pH 7.6 and ph 9.4. The binding of NAD+ to carboxymethylated enzyme at pH 7.6 and pH 9.4 also occurs without cooperativity. The possible implications of these findings for the involvement of ionising groups in the enzyme in the subunit interactions responsible for negative cooperativity, previously reported for coenzyme binding at pH 7.4--8.6, are discussed.     

Purification and characterization of D-glyceraldehyde-3-phosphate dehydrogenase from the thermophilic archaebacterium Methanothermus fervidus

The D-glyceraldehyde-3-phosphate dehydrogenase from the extremely thermophilic archaebacterium Methanothermus fervidus was purified and crystallized. The enzyme is a homomeric tetramer (molecular mass of subunits 45 kDa). Partial sequence analysis shows homology to the enzymes from eubacteria and from the cytoplasm of eukaryotes. Unlike these enzymes, the D-glyceraldehyde-3-phosphate dehydrogenase from Methanothermus fervidus reacts with both NAD+ and NADP+ and is not inhibited by pentalenolactone. The enzyme is intrinsically stable up to 75 degrees C. It is stabilized by the coenzyme NADP+ and at high ionic strength up to about 90 degrees C. Breaks in the Arrhenius and Van't Hoff plots indicate conformational changes of the enzyme at around 52 degrees C.     

Inactivation of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase by koningic acid

Koningic acid, a sesquiterpene antibiotic, is a specific inhibitor of the enzyme glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12). In the presence of 3 mM of NAD+, koningic acid irreversibly inactivated the enzyme in a time-dependent manner. The pseudo-first-order rate constant for inactivation (kapp) was dependent on koningic acid concentration in saturate manner, indicating koningic acid and enzyme formed a reversible complex prior to the formation of an inactive, irreversible complex; the inactivation rate (k 3) was 5.5.10(-2) s-1, with a dissociation constant for inactivation (Kinact) of 1.6 microM. The inhibition was competitive against glyceraldehyde 3-phosphate with a Ki of 1.1 microM, where the Km for glyceraldehyde 3-phosphate was 90 microM. Koningic acid inhibition was uncompetitive with respect to NAD+. The presence of NAD+ accelerated the inactivation. In its absence, the charcoal-treated NAD+-free enzyme showed a 220-fold decrease in apparent rate constant for inactivation, indicating that koningic acid sequentially binds to the enzyme next to NAD+. The enzyme, a tetramer, was inactivated when maximum two sulfhydryl groups, possibly cysteine residues at the active sites of the enzyme, were modified by the binding of koningic acid. These observations demonstrate that koningic acid is an active-site-directed inhibitor which reacts predominantly with the NAD+-enzyme complex.     

Glyceraldehyde 3-phosphate dehydrogenase of Scenedesmus obliquus: Promotion of NADPH-dependent activity and antagonisation by NAD

The glyceraldehyde 3-phosphate dehydrogenase activity of extracts from heterotrophic Scenedesmus obliquus was linked predominantly to NADH. However, on DEAE-cellulose chromatography the enzyme was eluted by a gradient of phosphate in a form characterized by high NADPH-dependent glyceraldehyde 3-phosphate dehydrogenase activity. This interconversion of enzyme forms could be prevented by the presence of NAD during DEAE-cellulose chromatography.High concentrations of phosphate stimulated the NADPH-dependent activity of the purified enzyme at the expense of activity linked to NADH and these changes were associated with depolymerization of a hexadecamer to a tetramer. The effect of phosphate on the rates of increase in NADPH-dependent activity and of a decrease in activity linked to NADH was cooperative with a Hill coefficient of 3.2. The inversely related changes in coenzyme specificity were inhibited to the same extent by NAD and the response to this ligand was anticooperative. These findings imply a strictly inverse proportional relationship between the rates of change of NADH and NADPH-linked activity. In the presence of dithiothreitol, low concentrations of phosphate promoted NADPH-dependent activity by stabilising the unstable tetrameric form produced from the hexadecamer by the thiol.These phenomena are discussed in relation to a general mechanism for the in vivo promotion of NADPH-dependent glyceraldehyde 3-phosphate dehydrogenase activity.     

Evidence for ligand-induced conformational changes in rabbit-muscle glyceraldehyde-3-phosphate dehydrogenase.

The tetrameric glyceraldehyde-3-phosphate dehydrogenase from rabbit muscle binds NAD+ and some of its analogues in a negatively cooperative manner, whereas other NAD+ analogues bind non-cooperatively to this enzyme. Subsequent to alkylation of a fraction of the active sites of the enzyme with the fluorescent SH reagent N-iodoacetyl-N'-(5-sulfo-1-naphthyl)-ethylenediamine, it was found that the alkylated sites bind NAD+ and NAD+ analogues with a markedly reduced affinity as compared with non-alkylated sites. It was therefore feasible to measure the fluorescence and the circular polarization of the luminescence of the enzyme-bound alkyl groups as a function of binding of NAD+ and of NAD+ analogues to the non-alkylated sites. The changes observed indicate that ligand binding to the non-alkylated sites induces changes in the fluorescence properties of the alkyl groups bound to neighbouring subunits, most likely through the protein moiety. The nature of these changes appears to depend on the structure of the coenzyme analogue. The binding of the non-cooperative binders acetyl-pyridine--adenine dinucleotide, ATP and ADP-ribose induce different conformational changes in the neighbouring vacant subunit, as monitored by the spectroscopic properties of the bound alkyl group. These results in conjunction with other data support the view that the negative cooperativity in NAD+ binding to glyceraldehyde-3-phosphate dehydrogenase results from ligand-induced conformational changes. Furthermore, these results further support the view that subtle structural changes in the coenzyme molecule determine the nature of the conformational changes induced within the enzyme tetramer.     

Characterization of two glyceraldehyde-3-phosphate dehydrogenase isoenzymes from the pentalenolactone producer Streptomyces arenae.

Pentalenolactone (PL) irreversibly inactivates the enzyme glyceraldehyde-3-phosphate dehydrogenase [D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating)] (EC 1.2.1.12) and thus is a potent inhibitor of glycolysis in both procaryotic and eucaryotic cells. We showed that PL-producing strain Streptomyces arenae TU469 contains a PL-insensitive glyceraldehyde-3-phosphate dehydrogenase under conditions of PL production. In complex media no PL production was observed, and a PL-sensitive glyceraldehyde-3-phosphate dehydrogenase, rather than the insensitive enzyme, could be detected. The enzymes had the same substrate specificity but different catalytic and molecular properties. The apparent Km values of the PL-insensitive and PL-sensitive enzymes for glyceraldehyde-3-phosphate were 100 and 250 microM, respectively, and the PL-sensitive enzyme was strongly inhibited by PL under conditions in which the PL-insensitive enzyme was not inhibited. The physical properties of the PL-insensitive enzyme suggest that the protein is an octamer, whereas the PL-sensitive enzyme, like other glyceraldehyde-3-phosphate dehydrogenases, appears to be a tetramer.     

Rabbit muscle D-glyceraldehyde-3-phosphate dehydrogenase: half-of-the-sites reactivity of the enzyme modified at arginine residues.

Modification of a single arginine residue per subunit of rabbit muscle apo-D-Glyceraldehyde-3-phosphate dehydrogenase does not affect the rate of hydrolysis of p-nitrophenyl acetate catalyzed by the enzyme, but locks the tetramer in a conformation wherein only two active sites are functioning. The modified enzyme also exhibits half-of-the sites reactivity towards iodoacetate and iodoacetamide. On the other hand, its NAD(+)-binding characteristics remain unchanged. Evidence is presented supporting the view that mechanisms of half-of-the-sites reactivity and negative cooperativity in coenzyme binding are different. The results are consistent with a built-in asymmetry of the tetramer and suggest that the arginine residue (probably Arg-231) controls the conformational transition between the asymmetric and symmetric states of the tetramer.     

Immobilized hybrids of glyceraldehyde-3-phosphate dehydrogenase.

Yeast glyceraldehyde-3-phosphate dehydrogenase (glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12) immobilized on CNBr-activated Sepharose 4-B has been subjected to dissociation to obtain matrix-bound dimeric species of the enzyme. Hybridization was then performed using soluble glyceraldehyde-3-phosphate dehydrogenase isolated from rat skeletal muscle. Immobilized hybrid tetramers thus obtained were demonstrated to exhibit two distinct pH-optima of activity characteristic of the yeast and muscle enzymes, respectively. The results indicate that under appropriate conditions the activity of each of the dimers composing the immobilized hybrid tetramer can be studied separately.     

Autonomous folding of the excised coenzyme-binding domain of D-glyceraldehyde 3-phosphate dehydrogenase from Thermotoga maritima.

An important question in protein folding is whether compact substructures or domains are autonomous units of folding and assembly. The protomer of the tetrameric D-glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima has a complex coenzyme-binding domain, in which residues 1-146 form a compact substructure with the last 31 residues (313-333). Here it is shown that the gene of a single-chain protein can be expressed in Escherichia coli after deleting the 163 codons corresponding to the interspersed catalytic domain (150-312). The purified gene product is a soluble, monomeric protein that binds both NAD+ and NADH strongly and possesses the same unfolding transition induced by guanidinium chloride as the native tetramer. The autonomous folding of the coenzyme-binding domain has interesting implications for the folding, assembly, function, and evolution of the native enzyme.     

Interaction of the dissociable glycerol-3-phosphate dehydrogenase and fructose-1,6-bisphosphate aldolase. Quantitative analysis by an extrinsic fluorescence probe

Cytoplasmic sn-glycerol-3-phosphate dehydrogenase, labelled covalently with fluorescein isothiocyanate, shows an enzyme-concentration-dependent fluorescence anisotropy. The anisotropy versus enzyme concentration curve is shifted towards higher concentrations when substrates are present. The comparison of the dissociation constants estimated from anisotropy measurements and derived from kinetic experiments suggests that the substrate-induced dissociation of the dimeric dehydrogenase is slow with respect to the enzymatic reaction catalyzed by either its monomeric or dimeric form. The fluorescence anisotropy of the fluorescent dye-labelled dehydrogenase increase with time upon addition of unlabelled fructose-1,6-bisphosphate aldolase approaching a limiting value. This fact indicates the binding of fructose-1,6-bisphosphate aldolase aldose aldolase to glycerolphosphate dehydrogenase. A model is proposed assuming simultaneous binding of tetrameric fructose-1,6-bisphosphate aldolase to monomeric and dimeric glycerolphosphate dehydrogenase with 1:1 stoichiometry. The dissociation constants, as parameters fitted to the experimental curves, were estimated as 0.2 microM and 1 microM for aldolase-dimeric-glycerolphosphate-dehydrogenase and aldolase-monomeric-glycerolphosphate-dehydrogenase complexes respectively.     

Antibodies to the nonnative forms of d-glyceraldehyde-3-phosphate dehydrogenase: identification, purification, and influence on the renaturation of the enzyme.

Monoclonal antibodies of two clones reacting with the nonnative forms of d-glyceraldehyde-3-phosphate dehydrogenase, EC 1.2.1.12 (GAPDH), were obtained. Antibodies of clone 6C5 belonged to IgG1 subtype; antibodies of clone 6G7 belonged to IgM type. The interaction of antibodies of both clones with the immobilized and soluble enzyme was studied. The specificity of antibodies to the definite oligomeric forms was demonstrated on immobilized monomers, dimers, and tetramers of GAPDH. The affinity of antibodies to monomeric and dimeric forms of GAPDH, either active or not, was demonstrated. At the same time the antibodies did not react with the tetrameric enzyme. The binding of antibodies had no influence on the enzymatic activity. However, the addition of antibodies to the denatured enzyme blocked the spontaneous renaturation of GAPDH. The immobilized antibodies of both clones were successfully used for the purification of GAPDH solution from the denatured admixtures.     

Studies of coenzyme binding to rabbit muscle glyceraldehyde-3-phoshate dehydrogenase.

The binding of NAD+, NADH and adenosine diphosphoribose (Ado-PP-Rib) to a stable, highly active and nucleotide-free preparation of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate: NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12) has been studied. All three nucleotides quench the protein fluorescence to the same extent when they bind to the enzyme, and this property has been used to measure the dissociation constants for the two high-affinity binding sites for the nucleotides. The results indicate negative interactions between, or non-identify of, these two binding sites, to which NAD+ and NADH bind with similar affinity. The binding of NAD+ to the enzyme has been studied by spectrophotometric titrations at 360 nm. It appears that the binding of NAD+ to each of the four subunits of the enzyme contributes equally to the intensity of this 'Racker' band. The dissociation constants associated with the binding of the third and fourth molecules of NAD+ estimated from such titrations confirm some previous estimates. The binding of NADH to the enzyme causes a decrease of intensity of the absorbance of the coenzyme at 340 nm, and the dissociation constants for binding of the third and fourth molecules of NADH have been estimated from spectrophotometric titrations. They are the same as those for NAD+. Judging by the apparent dissociation constants, negative interactions on binding the third molecule of NAD+ or NADH are more marked than those associated with the binding of the second and fourth molecules, suggesting that a major conformational change occurs at half-saturation of the tetramer with coenzyme.     

Catalytic mechanism and interactions of NAD+ with glyceraldehyde-3-phosphate dehydrogenase: correlation of EPR data and enzymatic studies

Perdeuterated spin label (DSL) analogs of NAD+, with the spin label attached at either the C8 or N6 position of the adenine ring, have been employed in an EPR investigation of models for negative cooperativity binding to tetrameric glyceraldehyde-3-phosphate dehydrogenase and conformational changes of the DSL-NAD+-enzyme complex during the catalytic reaction. C8-DSL-NAD+ and N6-DSL-NAD+ showed 80 and 45% of the activity of the native NAD+, respectively. Therefore, these spin-labeled compounds are very efficacious for investigations of the motional dynamics and catalytic mechanism of this dehydrogenase. Perdeuterated spin labels enhanced spectral sensitivity and resolution thereby enabling the simultaneous detection of spin-labeled NAD+ in three conditions: (1) DSL-NAD+ freely tumbling in the presence of, but not bound to, glyceraldehyde-3-phosphate dehydrogenase, (2) DSL-NAD+ tightly bound to enzyme subunits remote (58 A) from other NAD+ binding sites, and (3) DSL-NAD+ bound to adjacent monomers and exhibiting electron dipolar interactions (8-9 A or 12-13 A, depending on the analog). Determinations of relative amounts of DSL-NAD+ in these three environments and measurements of the binding constants, K1-K4, permitted characterization of the mathematical model describing the negative cooperativity in the binding of four NAD+ to glyceraldehyde-3-phosphate dehydrogenase. For enzyme crystallized from rabbit muscle, EPR results were found to be consistent with the ligand-induced sequential model and inconsistent with the pre-existing asymmetry models. The electron dipolar interaction observed between spin labels bound to two adjacent glyceraldehyde-3-phosphate dehydrogenase monomers (8-9 or 12-13 A) related by the R-axis provided a sensitive probe of conformational changes of the enzyme-DSL-NAD+ complex. When glyceraldehyde-3-phosphate was covalently bound to the active site cysteine-149, an increase in electron dipolar interaction was observed. This increase was consistent with a closer approximation of spin labels produced by steric interactions between the phosphoglyceryl residue and DSL-NAD+. Coenzyme reduction (DSL-NADH) or inactivation of the dehydrogenase by carboxymethylation of the active site cysteine-149 did not produce changes in the dipolar interactions or spatial separation of the spin labels attached to the adenine moiety of the NAD+. However, coenzyme reduction or carboxymethylation did alter the stoichiometry of binding and caused the release of approximately one loosely bound DSL-NAD+ from the enzyme. These findings suggest that ionic charge interactions are important in coenzyme binding at the active site.     

Catalytically Active Monomers of E. coli Glyceraldehyde-3-Phosphate Dehydrogenase

Monomeric forms of E. coli glyceraldehyde-3-phosphate dehydrogenase have been prepared using two different experimental approaches: (1) covalent immobilization of a tetramer on a solid support via a single subunit with subsequent dissociation of non-covalently bound subunits in the presence of urea, and (2) entrapment of monomeric species into reversed micelles of Aerosol OT in octane. Isolated monomers were shown to be catalytically active, exhibiting K _M values close to the parameters characteristic of the tetrameric forms. Like tetramers, isolated monomers did not use NADP7 as a coenzyme.     

Catalytically Active Monomers of E. coli Glyceraldehyde-3-Phosphate Dehydrogenase

Monomeric forms of E. coli glyceraldehyde-3-phosphate dehydrogenase have been prepared using two different experimental approaches: (1) covalent immobilization of a tetramer on a solid support via a single subunit with subsequent dissociation of non-covalently bound subunits in the presence of urea, and (2) entrapment of monomeric species into reversed micelles of Aerosol OT in octane. Isolated monomers were shown to be catalytically active, exhibiting K _M values close to the parameters characteristic of the tetrameric forms. Like tetramers, isolated monomers did not use NADP7 as a coenzyme.     

P but not R-axis interface is involved in cooperative binding of NAD on tetrameric phosphorylating glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus

Homotetrameric phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Bacillus stearothermophilus can be described as a dimer of dimers with three non-equivalent P, R, and Q interfaces. In our previous study, negative cooperativity in NAD binding to wild-type GAPDH was interpreted according to the induced-fit model in terms of two independent dimers with two interacting binding sites in each dimer. Two dimeric mutant GAPDHs, i.e. Y46G/S48G and D186G/E276G, were shown to exhibit positive cooperativity in NAD binding. Based on the molecular modeling of the substitutions and the fact that the most extensive inter-subunit interactions are formed across the P-axis interface of the tetramer, it was postulated that both dimeric mutant GAPDHs were of O-P type. Therefore, the P-axis interface was assumed to play a major role in causing cooperativity in NAD binding.Here, two other mutant GAPDHs, Y46G/R52G and D282G, have been studied. Using small angle X-ray scattering, the dimeric form of the D282G mutant GAPDH is shown to be of O-R type whereas both dimeric mutant GAPDHs Y46G/R52G and Y46G/S48G are of O-P type. Similarly to dimeric Y46G/S48G mutant GAPDH, the dimeric Y46G/R52G mutant GAPDH exhibits positive cooperativity in NAD binding. On the other hand, no significant cooperativity in NAD binding to the dimeric form of the D282G mutant GAPDH is observed, whereas its tetrameric counterpart exhibits negative cooperativity, similarly to the wild-type enzyme. Altogether, the results support the view that the P-axis interface is essential in causing cooperativity in NAD binding by transmitting the structural information induced upon cofactor binding from one subunit to the other one within O-P/Q-R dimers in contrast to the R-axis interface, which does not transmit structural information within O-R/Q-P dimers. The absence of activity of O-P and O-R dimer GAPDHs is the consequence of a pertubation of the conformation of the active site, at least of the nicotinamide subsite, as evidenced by the absence of an ion pair between catalytic residues C149 and H176 and the greater accessibility of C149 to a thiol kinetic probe.     

Dissociation and aggregation of D-glyceraldehyde-3-phosphate dehydrogenase during denaturation by guanidine hydrochloride

The inactivation of lobster muscle D-glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12) (GAPDH) during guanidine hydrochloride (GdnHCl) denaturation has been compared with its state of aggregation and unfolding, by light scattering and fluorescence measurements. The enzyme first dissociates at low concentrations of GdnHCl, followed by the formation of a highly aggregated state with increasing denaturant concentrations, and eventually by complete unfolding and dissociation to the monomer at concentrations of greater than 2 M GdnHCl. The aggregation and final dissociation correspond roughly with the two stages of fluorescence changes reported previously (Xie, G.-F. and Tsou, C.-L. (1987) Biochim. Biophys. Acta 911, 19-24). Rate measurements show a very rapid inactivation, the extents of which increase with increasing concentrations of GdnHCl. This initial rapid phase of inactivation which takes place before dissociation and unfolding of the molecule is in agreement with the results obtained with other enzymes, that the active site is affected before noticeable conformational changes can be detected for the enzyme molecule as a whole. A scheme for the steps leading to the final denaturation, and dissociation of the enzyme to the inactive and unfolded monomer, is proposed.