Diffusion-controlled and concerted base catalysis in the decomposition of hemithioacetals

Barnett, Ronald E.; Jencks, William P.

doi:10.1021/ja01052a038

Cited by 58 publications

(41 citation statements)

References 0 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…They conclude that under their conditions NAD+ and glyceraldehyde 3-phosphate bind to the apoenzyme in a random order. At this high pH reactions associated with hemithioacetal formation and dissociation would be rapid for a typical alkyl thiol of pK 9 (Barnett & Jencks, 1969), so that the rate constants of the reversible reaction of glyceraldehyde 3-phosphate and the apoenzyme may be larger than the catalytic-centre activity. This serves to illustrate that the reaction scheme is only designed to represent the major reaction pathway of the glyceraldehyde 3-phosphate dehydrogenase mechanism especially at physiological pH.…”

Section: Discussionmentioning

confidence: 99%

Rate-determining processes and the number of simultaneously active sites of d-glyceraldehyde 3-phosphate dehydrogenase

Trentham

1971

Biochemical Journal

View full text Add to dashboard Cite

Transient kinetic studies of the reversible oxidative phosphorylation of d-glyceraldehyde 3-phosphate catalysed by d-glyceraldehyde 3-phosphate dehydrogenase show that all four sites of the tetrameric lobster enzyme are simultaneously active, apparently with equal reactivity. The rate-determining step of the oxidative phosphorylation is NADH release at high pH and phosphorolysis of the acyl-enzyme at low pH. For the reverse reaction the rate-determining step is a process associated with NADH binding, probably a conformation change, at high pH and d-glyceraldehyde 3-phosphate release at low pH. NADH has previously been shown to be a competitive inhibitor of the enzyme with respect to d-glyceraldehyde 3-phosphate and vice versa. This is consistent with the mechanism deduced from transient experiments given the additional proviso that 1-arseno-3-phosphoglycerate has a half-life of about 1min or longer at pH7. The dissociation constants of d-glyceraldehyde 3-phosphate and 1,3-diphosphoglycerate to the NAD+-bound enzyme are too large to measure but are nevertheless consistent with the low Km values of these substrates.

show abstract

Section: Discussionmentioning

confidence: 99%

Rate-determining processes and the number of simultaneously active sites of d-glyceraldehyde 3-phosphate dehydrogenase

Trentham

1971

Biochemical Journal

View full text Add to dashboard Cite

show abstract

“…The decomposition of hemithioacetals occurs largely as a second-order process involving catalysis by OH-ion, and is diffusion-controlled for thiols with pK below 8. For more basic thiols the rate decreases with increasing basicity (Barnett & Jencks, 1969). Thus the OH--catalysed decomposition of the hemithioacetal of methyl thioglycollate (pK= 7.8) proceeds at least 10 times as fast as that for the hemithioacetal of J-mercaptoethanol (pK= 9.5).…”

Section: Effect Ofthiol On Apparent Aldehyde Concentrationmentioning

confidence: 99%

The reactions of D-glyceraldehyde 3-phosphate with thiols and the holoenzyme of D-glyceraldehyde 3-phosphate dehydrogenase and of inorganic phosphate with the acyl-holoenzyme

Armstrong

Trentham

1976

Biochemical Journal

View full text Add to dashboard Cite

D-Glyceraldehyde 3-phosphate forms adducts with thiols. These adducts, which are presumed to be hemithioacetals, equilibrate rapidly with the unhydrated form of the aldehyde, which is the subtrate for D-glyceraldehyde 3-phosphate dehydrogenase. The adduct provides a substrate buffer system whereby a constant low free aldehyde concentration can be maintained during the oxidation of aldehyde by the enzyme and NAD+. With this system, the kinetics of the association of the aldehyde with the enzyme were examined. The rate profile for this reaction is a single exponential process, showing that all four active sites of the enzyme have equivalent and independent reactivity towards the aldehyde, with an apparent second-order rate constant of 5 X 10(7)M-1-S-1 at pH8.0 and 21 degrees C. The second-order rate constant becomes 8 X 10(7)M-1-S-1 when account is taken of the forward and reverse catalytic rate constants of the dehydrogenase. The pH-dependence of the observed rate constant is consistent with a requirement for the unprotonated form of a group of pK 6.1, which is the pK observed for second ionization of glyceraldehyde 3-phosphate. The rate of phosphorolysis of the acyl-enzyme intermediate during the steady-state oxidative phosphorylation of the aldehyde was studied, and is proportional to the total Pi concentration up to at least 1 mM-Pi at pH 7.5. The pH-dependence of the rate of NADH generation under these conditions can be explained by the rate law d[NADA]/dt = k[acy] holoenzyme][PO4(3-)-A1, where thioester bond, although kinetically indistinguishable rate equations for the reaction are possible. The rates of the phosphorolysis reaction and of the aldehyde-association reaction decrease with increasing ionic strength, suggesting that the active site of the enzyme has cationic groups which are involved in the reaction of the enzyme with anionic substrates.

show abstract

“…8 sec, measurements of somewhat faster rate processes are by no means impossible. Reaction halftimes of 0.5 msec were measured with a stopped-flow having a dead time of 2 msec (Pearlmutter and Stuehr, 1968), and in an even more impressive case, Barnett and Jencks (1969) were able to measure half-times as short as 0.7 msec, with a stopped-flow having a dead time of ca. 5 msec.…”

Section: O O P E R a T I V E I O N B I N D I N G T O T R N Amentioning

confidence: 99%

Cooperative binding of magnesium to transfer ribonucleic acid studied by a fluorescent probe

Lynch¹,

Schimmel²

1974

Biochemistry

102

View full text Add to dashboard Cite

The mechanism of cooperative binding of Mg2+ to tRNA at pH 7.5 has been studied by means of a naphthyl fluorescent probe covalently attached to the isoleucyl amino moiety of isoleucyl-tRNA1le. The emission of the probe is remarkably sensitive to Mg2+ concentration. This sensitivity is due to structural changes in the tRNA induced by binding of Mg2+ to "cooperative" sites, as shown by several criteria. Furthermore, it is demonstrated that the probe itself simply monitors but does not cause the structural changes that are observed. The time dependence of the emission changes associated with Mg2+ additions to Mg2+-free tRNA has been carefully investigated. The emission changes are biphasic,

show abstract

Diffusion-controlled and concerted base catalysis in the decomposition of hemithioacetals

Cited by 58 publications

References 0 publications

Rate-determining processes and the number of simultaneously active sites of d-glyceraldehyde 3-phosphate dehydrogenase

Rate-determining processes and the number of simultaneously active sites of d-glyceraldehyde 3-phosphate dehydrogenase

The reactions of D-glyceraldehyde 3-phosphate with thiols and the holoenzyme of D-glyceraldehyde 3-phosphate dehydrogenase and of inorganic phosphate with the acyl-holoenzyme

Cooperative binding of magnesium to transfer ribonucleic acid studied by a fluorescent probe

Contact Info

Product

Resources

About