scholarly journals Using pre-steady state and steady-state kinetics to examine the roles of active site residues in L-DOPA dioxygenase

2020 ◽  
Author(s):  
Keri L Colabroy
Keyword(s):  
Steady State ◽  
Active Site ◽  
Active Site Residues ◽  
Steady State Kinetics

scholarly journals Catalytic and structural contributions for glutathione-binding residues in a Delta class glutathione S-transferase

2004 ◽  
Vol 382 (2) ◽  
pp. 751-757 ◽  
Author(s):  
Pakorn WINAYANUWATTIKUN ◽  
Albert J. KETTERMAN
Keyword(s):  
Binding Site ◽  
Active Site ◽  
Structural Integrity ◽  
Catalytic Mechanism ◽  
Tertiary Structure ◽  
Site Directed Mutagenesis ◽  
Active Site Residues ◽  
Anopheles Dirus ◽  
Steady State Kinetics ◽  
Cellular Detoxification

Glutathione S-transferases (GSTs) are dimeric proteins that play a major role in cellular detoxification. The GSTs in mosquito Anopheles dirus species B, an important malaria vector in South East Asia, are of interest because they can play an important role in insecticide resistance. In the present study, we characterized the Anopheles dirus (Ad)GST D3-3 which is an alternatively spliced product of the adgst1AS1 gene. The data from the crystal structure of GST D3-3 shows that Ile-52, Glu-64, Ser-65, Arg-66 and Met-101 interact directly with glutathione. To study the active-site function of these residues, alanine substitution site-directed mutagenesis was performed resulting in five mutants: I52A (Ile-52→Ala), E64A, S65A, R66A and M101A. Interestingly, the E64A mutant was expressed in Escherichia coli in inclusion bodies, suggesting that this residue is involved with the tertiary structure or folding property of this enzyme. However, the I52A, S65A, R66A and M101A mutants were purified by glutathione affinity chromatography and the enzyme activity characterized. On the basis of steady-state kinetics, difference spectroscopy, unfolding and refolding studies, it was concluded that these residues: (1) contribute to the affinity of the GSH-binding site (‘G-site’) for GSH, (2) influence GSH thiol ionization, (3) participate in kcat regulation by affecting the rate-limiting step of the reaction, and in the case of Ile-52 and Arg-66, influenced structural integrity and/or folding of the enzyme. The structural perturbations from these mutants are probably transmitted to the hydrophobic-substrate-binding site (‘H-site’) through changes in active site topology or through effects on GSH orientation. Therefore these active site residues appear to contribute to various steps in the catalytic mechanism, as well as having an influence on the packing of the protein.

scholarly journals Roles of active-site residues in catalysis, substrate binding, cooperativity, and the reaction mechanism of the quinoprotein glycine oxidase

2020 ◽  
Vol 295 (19) ◽  
pp. 6472-6481
Author(s):  
Kyle J. Mamounis ◽  
Erik T. Yukl ◽  
Victor L. Davidson
Keyword(s):  
Active Site ◽  
Protein Function ◽  
Marine Bacterium ◽  
Substrate Binding ◽  
Active Site Residues ◽  
X Ray Crystallography ◽  
Steady State Kinetics ◽  
Hill Coefficients ◽  
The Hill ◽  
Glycine Oxidase

The quinoprotein glycine oxidase from the marine bacterium Pseudoalteromonas luteoviolacea (PlGoxA) uses a protein-derived cysteine tryptophylquinone (CTQ) cofactor to catalyze conversion of glycine to glyoxylate and ammonia. This homotetrameric enzyme exhibits strong cooperativity toward glycine binding. It is a good model for studying enzyme kinetics and cooperativity, specifically for being able to separate those aspects of protein function through directed mutagenesis. Variant proteins were generated with mutations in four active-site residues, Phe-316, His-583, Tyr-766, and His-767. Structures for glycine-soaked crystals were obtained for each. Different mutations had differential effects on kcat and K0.5 for catalysis, K0.5 for substrate binding, and the Hill coefficients describing the steady-state kinetics or substrate binding. Phe-316 and Tyr-766 variants retained catalytic activity, albeit with altered kinetics and cooperativity. Substitutions of His-583 revealed that it is essential for glycine binding, and the structure of H583C PlGoxA had no active-site glycine present in glycine-soaked crystals. The structure of H767A PlGoxA revealed a previously undetected reaction intermediate, a carbinolamine product-reduced CTQ adduct, and exhibited only negligible activity. The results of these experiments, as well as those with the native enzyme and previous variants, enabled construction of a detailed mechanism for the reductive half-reaction of glycine oxidation. This proposed mechanism includes three discrete reaction intermediates that are covalently bound to CTQ during the reaction, two of which have now been structurally characterized by X-ray crystallography.

Nitrogen and Sulfur Transferases

2007 ◽  
Author(s):  
Perry A. Frey ◽  
Adrian D. Hegeman
Keyword(s):  
Steady State ◽  
Reaction Mechanism ◽  
Active Site ◽  
Reaction Mechanisms ◽  
Radical Reaction ◽  
Amino Group ◽  
Steady State Kinetics ◽  
Ping Pong ◽  
Polar Reaction

Unlike other group transfer reactions in biochemistry, the actions of nitrogen transferring enzymes do not follow a single unifying chemical principle. Nitrogen-transferring enzymes catalyze aminotransfer, amidotransfer, and amidinotransfer. An aminotransferase catalyzes the transfer of the NH2 group from a primary amine to a ketone or aldehyde. An amidotransferase catalyzes the transfer of the anide-NH2 group from glutamine to another group. These reactions proceed by polar reaction mechanisms. Aminomutases catalyze 1,2-intramolecular aminotransfer, in which an amino group is inserted into an adjacent C—H bond. The action of lysine 2,3-aminomutase, described in chapter 7, is an example of an aminomutase that functions by a radical reaction mechanism. Tyrosine 2,3-aminomutase also catalyzes the 2,3-amino migration, but it does so by a polar reaction mechanism. In this chapter, we consider NH2-transferring enzymes that function by polar reaction mechanisms. Transaminases or aminotransferases are the most extensively studied pyridoxal-5'-phosphate (PLP)–dependent enzymes, and many aminotransferases catalyze essential steps in catabolic and anabolic metabolism. In the classic transaminase reaction, aspartate aminotransferase (AAT) catalyzes the fully reversible reaction of L-aspartate with α-ketoglutarate according to fig. 13-1 to form oxaloacetate and L-glutamate. Like all aminotransferases, AAT is PLP dependent, and PLP functions in its classic role of providing a reactive carbonyl group to function in facilitating the cleavage of the α-H of aspartate and the departure of the α-amino group of aspartate for transfer to α-ketoglutarate (Snell, 1962). PLP in the holoenzyme functions in essence to stabilize the α-carbanions of L-aspartate or L-glutamate, the major biological role of PLP discussed in chapter 3. The functional groups of the enzyme catalyze steps in the mechanism, such as the 1,3-prototropic shift of the α-proton to C4' of pyridoxamine 5'-phosphate (PMP). The steady-state kinetics corresponds to the ping pong bi bi mechanism shown at the bottom of fig. 13-1. This mechanism allows L-aspartate to react with the internal aldimine, E=PLP in fig. 13-1, to produce an equivalent of oxaloacetate, with conversion of PLP to PMP at the active site (E.PMP), the free, covalently modified enzyme in the ping pong mechanism.

scholarly journals Steady-state kinetics and inhibition of anaerobically purified human homogentisate 1,2-dioxygenase

2005 ◽  
Vol 386 (2) ◽  
pp. 305-314 ◽  
Author(s):  
Edwin J. A. VELDHUIZEN ◽  
Frédéric H. VAILLANCOURT ◽  
Cheryl J. WHITING ◽  
Marvin M.-Y. HSIAO ◽  
Geneviève GINGRAS ◽  
...  
Keyword(s):  
Steady State ◽  
Active Site ◽  
Ternary Complex ◽  
Specific Activity ◽  
Complex Mechanism ◽  
Substrate Analogues ◽  
Steady State Kinetics ◽  
Km Value ◽  
Substrate Concentrations ◽  
Active Preparation

HGO (homogentisate 1,2-dioxygenase; EC 1.13.11.5) catalyses the O2-dependent cleavage of HGA (homogentisate) to maleylacetoacetate in the catabolism of tyrosine. Anaerobic purification of heterologously expressed Fe(II)-containing human HGO yielded an enzyme preparation with a specific activity of 28.3± 0.6 μmol·min−1·mg−1 (20 mM Mes, 80 mM NaCl, pH 6.2, 25 °C), which is almost twice that of the most active preparation described to date. Moreover, the addition of reducing agents or other additives did not increase the specific activity, in contrast with previous reports. The apparent specificity of HGO for HGA was highest at pH 6.2 and the steady-state cleavage of HGA fit a compulsory-order ternary-complex mechanism (Km value of 28.6±6.2 μM for HGA, Km value of 1240±160 μM for O2). Free HGO was subject to inactivation in the presence of O2 and during the steady-state cleavage of HGA. Both cases involved the oxidation of the active site Fe(II). 3-Cl HGA, a potential inhibitor of HGO, and its isosteric analogue, 3-Me HGO, were synthesized. At saturating substrate concentrations, HGO cleaved 3-Me and 3-Cl HGA 10 and 100 times slower than HGA respectively. The apparent specificity of HGO for HGA was approx. two orders of magnitude higher than for either 3-Me or 3-Cl HGA. Interestingly, 3-Cl HGA inactivated HGO only twice as rapidly as HGA. This contrasts with what has been observed in mechanistically related dioxygenases, which are rapidly inactivated by chlorinated substrate analogues, such as 3-hydroxyanthranilate dioxygenase by 4-Cl 3-hydroxyanthranilate.

scholarly journals DNA polymerase β: analysis of the contributions of tyrosine-271 and asparagine-279 to substrate specificity and fidelity of DNA replication by pre-steady-state kinetics

1997 ◽  
Vol 323 (1) ◽  
pp. 103-111 ◽  
Author(s):  
Vadim S. KRAYNOV ◽  
Brian G. WERNEBURG ◽  
Xuejun ZHONG ◽  
Hui LEE ◽  
Jinwoo AHN ◽  
...  
Keyword(s):  
Steady State ◽  
Substrate Specificity ◽  
Dna Polymerase ◽  
Excision Repair ◽  
Catalytic Efficiency ◽  
Main Function ◽  
Wild Type ◽  
Active Site Residues ◽  
Dna Polymerase Β ◽  
Steady State Kinetics

DNA polymerase β (pol β) from rat brain, overexpressed in Escherichia coli, was used as a model to study the factors responsible for substrate specificity [kpol, Kd(app) and kpol/Kd (app)] and fidelity during DNA synthesis. The roles of two active-site residues, Asn-279 and Tyr-271, were examined by construction of N279A, N279Q, Y271A, Y271F and Y271S mutants followed by structural analyses by NMR and CD and functional analyses by pre-steady-state kinetics. The results are summarized as follows. (i) None of the two-dimensional NMR spectra of the mutants was significantly perturbed relative to that for wild-type pol β, suggesting that Tyr-271 and Asn-279 are not important for the global structure of the protein. (ii) CD analyses of guanidinium hydrochloride-induced denaturation showed that all mutants behaved similarly to the wild type in the free energy of denaturation, suggesting that Tyr-271 and Asn-279 are not critical for the conformational stability of pol β. (iii) The Kd(app) for the correct dNTP was lower than that for the incorrect dNTP by a factor of 10-30 in the case of wild-type pol β. Upon mutation to give N279A and N279Q, the Kd(app) for the correct dNTP increased by a factor of 15-25. As a consequence, the Kd(app) values for the correct and incorrect nucleotides were similar for N279A and N279Q, suggesting that the main function of the side chain of Asn-279 is in discrimination between the binding of correct and incorrect dNTPs. (iv) In the case of the Y271A mutant, the fidelity and the catalytic efficiency kpol/Kd(app) were little perturbed relative to the wild type. However, both the kpol and Kd(app) values for dNTP were 4-8 times lower in the case of the Y271A mutant than the corresponding values for wild-type pol β. Since the chemical step may not be rate-limiting for wild-type pol β, the effect on kpol could be quite significant if it is caused by a perturbation in the chemical step. (v) Pol β displayed the greatest specificity towards the G:C base pair, which is incorporated during base excision repair of G:U and G:T mispairs. This specificity was slightly enhanced for the Y271F mutant.

scholarly journals The effects of hydrogen ion concentration on the simplest steady-state enzyme systems

1971 ◽  
Vol 123 (3) ◽  
pp. 445-453 ◽  
Author(s):  
P. Ottolenghi
Keyword(s):  
Steady State ◽  
Active Site ◽  
Hydrogen Ion ◽  
Ion Concentration ◽  
Hydrogen Ion Concentration ◽  
Substrate Complex ◽  
Perturbation Term ◽  
Enzyme Substrate ◽  
Enzyme Systems ◽  
Steady State Kinetics

Laidler (1955) showed that consideration of the effect of pH on enzymic mechanisms that obey steady-state kinetics leads to the inclusion in the equations of a ‘perturbation term’ that can introduce curvature into the Lineweaver–Burk plots. He also stated conditions in which this term vanishes. This term can lead to apparent activation by substrate. Further, several cases are shown in which simplification, but not disappearance, of the perturbation term can lead to linearity of Lineweaver–Burk plots. These cases arise when the ionization of groups at the active site either is unaffected or is completely prevented when the enzyme–substrate complex is formed. It is also shown that V(app.) can vary with pH without a concomitant change in Km(app.) in certain cases that obey steady-state kinetics without implying that Km=Ks. When the perturbation term is significant, Dixon's (1953) rules for the calculation of pK values will not always apply.

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