The fluorometric titration of GPD with NAD$\sp+$ has long been used as a convenient method for monitoring the binding of NAD$\sp+.$ A reverse fluorometric titration was developed to minimize the disadvantages involved in the conventional fluorometric titration such as inner filter effect, physical perturbation of samples, and the correction for dilution of NAD$\sp+.$ The protein was initially saturated by NAD$\sp+,$ then the intrinsic protein fluorescence increase was followed as NAD$\sp+$ was removed by phosphodiesterase (PDE) or NADase. The time dependent increase in protein fluorescence was then correlated with the decrease in NAD$\sp+$ concentration as determined by independent measurement of the kinetics of the PDE (or NADase) reaction. The method was used to evaluate the binding of NAD$\sp+$ to yeast glyceraldehyde 3-phosphate dehydrogenase (GPD) and the K$\sb{\rm d}$s are in good agreement with the values obtained by more conventional methods (such as equilibrium dialysis and ultrafiltration) The method was used to study the effect of modification of the Cys-149 thiols on the GPD affinity for NAD$\sp{+}$. The alkylating groups, no matter their sizes and charges, have a similar influence on the coenzyme binding site ($\sim$100 fold weakened). Phosphate and phosphonate substituents on the right position of the modifiers can reduce the effect of alkylation on the enzyme NAD$\sp+$ binding sites. Glycidol phosphate and bromoacetylphosphonate (BAP) modified GPDs have higher affinities for NAD$\sp+$ than the other alkylated GPDs. Phosphoglyceroyl GPD (only diacylated) has no appreciable difference from native GPD in NAD$\sp+$ binding. The effect of modification of the active-site thiols on the protein lifetime and denaturation by Gdn$\cdot$HCl were examined. The significance of these effects on the enzyme catalytic action is discussed How a metabolite (such as 1,3-BPG) transfer between pairs of enzyme catalyzing consecutive reactions (as GPD and PGK in glycolysis) is controversial. To further explore this situation the effect of PGK on the G3P oxidative phosphorolysis (or arsenorylsis), and on the relaxation of the equilibrium transposition was examined (by adding TIM to shift the equilibrium of GPD reaction toward the reverse direction). In contrast to the higher equilibrium level of NADH expected in the presence of PGK, the (NADH) $\sb{\rm eq}$ was even lower than that in the absence of PGK. The rate of NADH disappearance following the addition of TIM to the pseudo-equilibrium system was reduced in the presence of PGK. The effect of PGK on these reactions cannot be explained if PGK only acts as a competitor of GPD for the substrate (G3P). These effects are readily interpreted in terms of a ternary 1,3-BPG$\cdot$PGK$\cdot$GPD complex which has been shown to undergo reverse reaction more slowly than does 1,3-BPG$\cdot$GPD complex