The kinetics of enzyme-catalyzed reactions with two or more substrates or products☆II. Inhibition: Nomenclature and theory

Ww, Cleland

doi:10.1016/0006-3002(63)91815-8

Cited by 299 publications

(127 citation statements)

References 11 publications

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“…The obtained results permit us, according to the considerations of Cleland [41], to rule out a ping-pong mechanism and are consistent with a sequential mechanism. A sequential mechanism has been proposed for PNP from both high-and low-molecular mass class [23], whereas no evidence for a ping-pong mechanism has been reported.…”

Section: Mechanism Of the Reactionsupporting

confidence: 71%

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Methylthioadenosine phosphorylase from the archaeon Pyrococcus furiosus

Cacciapuoti¹,

Moretti

Forte³

et al. 2004

European Journal of Biochemistry

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The extremely heat-stable 5¢-methylthioadenosine phosphorylase from the hyperthermophilic archaeon Pyrococcus furiosus was cloned, expressed to high levels in Escherichia coli, and purified to homogeneity by heat precipitation and affinity chromatography. The recombinant enzyme was subjected to a kinetic analysis including initial velocity and product inhibition studies. The reaction follows an ordered Bi-Bi mechanism and phosphate binding precedes nucleoside binding in the phosphorolytic direction. 5¢-Methylthioadenosine phosphorylase from Pyrococcus furiosus is a hexameric protein with five cysteine residues per subunit. Analysis of the fragments obtained after digestion of the protein alkylated without previous reduction identified two intrasubunit disulfide bridges. The enzyme is very resistant to chemical denaturation and the transition midpoint for guanidinium chloride-induced unfolding was determined to be 3.0 M after 22 h incubation. This value decreases to 2.0 M in the presence of 30 mM dithiothreitol, furnishing evidence that disulfide bonds are needed for protein stability. The guanidinium chloride-induced unfolding is completely reversible as demonstrated by the analysis of the refolding process by activity assays, fluorescence measurements and SDS/PAGE. The finding of multiple disulfide bridges in 5¢-methylthioadenosine phosphorylase from Pyrococcus furiosus argues strongly that disulfide bond formation may be a significant molecular strategy for stabilizing intracellular hyperthermophilic proteins.Keywords: disulfide bonds; hyperthermostability; 5¢-methyl thioadenosine phosphorylase; purine nucleoside phosphorylase; Pyrococcus furiosus.Hyperthermophilic enzymes which retain their structure and function near the boiling point of water have been, over the past decade, the object of extensive studies on protein stabilization, folding and evolutionary aspects [1][2][3][4]. Moreover, their unique structure-function properties of high thermostability are potentially significant for developing biotechnological applications [5,6]. Thus, there is a great deal of interest in studies on the biochemical adaptation of hyperthermophiles whose enzymes provide unique models for the study and understanding of the evolution of enzymes in terms of structure, specificity and catalytic properties.Much work has been done to identify the structural determinants of the enhanced stability of hyperthermophilic proteins. Several mechanisms of thermal stabilization have been proposed, among which additional networks of salt bridges and hydrogen bonds, improved packing density and enhanced secondary structure are the most cited [2][3][4][7][8][9]. In spite of this, no general rules have been established to date, and it has been concluded that each protein evolves individually through a limited number of factors that occur at different levels, also involving the amino acid sequence and the quaternary structure of the proteins.In recent years, growing attention has been paid to the presence of disulfide bonds in intracellular hy...

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Section: Mechanism Of the Reactionsupporting

confidence: 71%

“…From the secondary plots of slopes and intercepts vs. the concentrations of inhibitors, shown as insets of Fig. 3, an inhibition constant can be determined from the slope replot (K is ) or the intercept replot (K ii ) and is the horizontal intercept on the plot [41].…”

Section: Mechanism Of the Reactionmentioning

confidence: 99%

Methylthioadenosine phosphorylase from the archaeon Pyrococcus furiosus

Cacciapuoti¹,

Moretti

Forte³

et al. 2004

European Journal of Biochemistry

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“…Replots of the slopes and y-intercepts of the LB plot vs. I were analyzed to classify the inhibition type according to Cleland (1963). In case of doubts on the inhibi tion type, additional plots were analyzed and com pared with the literature (Keleti, 1986), i.e.…”

Section: A Linear Regressionmentioning

confidence: 99%

Synthesis of 6-Aryloxy- and 6-Arylalkoxy-2-chloropurines and Their Interactions with Purine Nucleoside Phosphorylase from Escherichia coli

Bzowska

Magnowska

Kazimierczuk³

1999

Zeitschrift Für Naturforschung C

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The phase transfer method was applied to perform the nucleophilic substitution of 2,6-dichloropurines by modified arylalkyl alcohol or phenols. Since under these conditions only the 6-halogen is exchanged, this method gives 2-chloro-6-aryloxy-and 2-chloro-6-arylalkoxypurines. 2-Chloro-6-benzylthiopurine was synthesized by alkylation of 2-chloro-6-thiopurine with benzyl bromide. The stereoisom ers of 2-chloro-6-(l-phenyl-l-ethoxy)purine were ob tained from R-and 5-enantiomers of sec.-phenylethylalcohol and 2,6-dichloropurine.A ll derivatives were tested for inhibition with purified hexameric E. coli purine nucleoside phosphorylase (PNP). For analogues showing IC50 < 10 ^.m, the type o f inhibition and inhibi tion constants were determined. In all cases the experimental data were best described by the mixed-type inhibition model and the uncompetitive inhibition constant, Kiu, was found to be several-fold lower than the competitive inhibition constant, Kic. This effect seems to be due to the 6-aryloxy-or 6-arylalkoxy substituent, because a natural PNP substrate adenine, as well as 2-chloroadenine, show mixed type inhibition with almost the same inhibition con stants Kiu and K1C . = 0.4, 0.6, 1.4, 1.4 and 2.2 [im, respectively). The i?-stereoisomer o f 2-chloro-6-(lpheny-l-ethoxy)purine has Kiu = 2.0 ^im, whereas inhibition o f its S counterpart is rather weak (IC50> 12 jim). More rigid (e.g. phenoxy-), non-planar (cyclohexyloxy-), or more bulky (2,4,6-trimethylphenoxy-) substituents at position 6 of the purine base gave less potent inhibi tors (IC50 = 26, 56 and >100 [im, respectively). The derivatives are selective inhibitors of hexameric "high-molecular mass" PNPs because no inhibitory activity vs. trimeric Cellulomonas sp. PNP was detected. The most potent inhibition was observed for 6-benzylthio-2-chloro-, 6-benzyloxy-2-chloro-, 2-chloro-6-(2-phenyl-l-ethoxy), 2-chloro-6-(3-phenyl-l-propoxy)-and 2-chloro-6-ethoxypurines (KiuBy establishing the ligand-dependent stabilization pattern of the E. coli PNP it was shown that the new derivatives, similarly as the natural purine bases, are able to form a dead-end ternary complex with the enzyme and orthophosphate. It was also shown that the derivatives are substrates in the reverse synthetic direction catalyzed by E. coli PNP

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“…The second substrate then interacts with the substituted enzyme intermediate to form the second product and regenerate the native enzyme. Ping-Pong and sequential mechanisms can be differentiated by steady-state kinetic analysis of the reaction using the procedures described by Cleland [45,46]. shown).…”

Section: Steady-state Rate Equation and Kinetic Parameters Of Rptp-camentioning

confidence: 99%

Steady-state kinetics of <i>Roystonea regia</i> palm tree peroxidase

Zamorano¹,

Cuadrado²,

Galende³

et al. 2012

JBPC

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The kinetics of enzyme-catalyzed reactions with two or more substrates or products☆II. Inhibition: Nomenclature and theory

Cited by 299 publications

References 11 publications

Methylthioadenosine phosphorylase from the archaeon Pyrococcus furiosus

Methylthioadenosine phosphorylase from the archaeon Pyrococcus furiosus

Synthesis of 6-Aryloxy- and 6-Arylalkoxy-2-chloropurines and Their Interactions with Purine Nucleoside Phosphorylase from Escherichia coli

Steady-state kinetics of <i>Roystonea regia</i> palm tree peroxidase

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The kinetics of enzyme-catalyzed reactions with two or more substrates or products☆II. Inhibition: Nomenclature and theory

Cited by 299 publications

References 11 publications

Methylthioadenosine phosphorylase from the archaeon Pyrococcus furiosus

Methylthioadenosine phosphorylase from the archaeon Pyrococcus furiosus

Synthesis of 6-Aryloxy- and 6-Arylalkoxy-2-chloropurines and Their Interactions with Purine Nucleoside Phosphorylase from Escherichia coli

Steady-state kinetics of &lt;i&gt;Roystonea regia&lt;/i&gt; palm tree peroxidase

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Steady-state kinetics of <i>Roystonea regia</i> palm tree peroxidase