Human liver aldehyde reductase: pH dependence of steady-state kinetic parameters

To obtain information on the reaction specificity of cystalysin from the spirochaete bacterium Treponema denticola, the interaction with l- and d-alanine has been investigated. Binding of both alanine enantiomers leads to the appearance of an external aldimine absorbing at 429nm and of a band absorbing at 498nm, indicative of a quinonoid species. Racemization and transamination reactions were observed to occur with both alanine isomers as substrates. The steady-state kinetic parameters for racemization, kcat and Km, for l-alanine are 1.05±0.03s−1 and 10±1mM respectively, whereas those for d-alanine are 1.4±0.1s−1 and 10±1mM. During the reaction of cystalysin with l- or d-alanine, a time-dependent loss of β-elimination activity occurs concomitantly with the conversion of the pyridoxal 5′-phosphate (PLP) coenzyme into pyridoxamine 5′-phosphate (PMP). The catalytic efficiency of the half-transamination of l-alanine is found to be 5.3×10−5 mM−1·s−1, 5-fold higher when compared with that of d-alanine. The partition ratio between racemization and half-transamination reactions is 2.3×103 for l-alanine and 1.4×104 for d-alanine. The pH dependence of the kinetic parameters for both the reactions shows that the enzyme possesses a single ionizing residue with pK values of 6.5–6.6, which must be unprotonated for catalysis. Addition of pyruvate converts the PMP form of the enzyme back into the PLP form and causes the concomitant recovery of β-elimination activity. In contrast with other PLP enzymes studied so far, but similar to alanine racemases, the apoform of the enzyme abstracted tritium from C4′ of both (4′S)- and (4′R)-[4′-3H]PMP in the presence of pyruvate. Together with molecular modelling of the putative binding sites of l- and d-alanine at the active site of the enzyme, the implications of these studies for the mechanisms of the side reactions catalysed by cystalysin are discussed.

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Influenza virus sialidase: effect of calcium on steady-state kinetic parameters

Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology ◽

10.1016/0167-4838(91)90526-6 ◽

1991 ◽

Vol 1077 (1) ◽

pp. 65-71 ◽

Cited By ~ 41

Author(s):

Andrew K.J. Chong ◽

Michael S. Pegg ◽

Mark von Itzstein

Keyword(s):

Influenza Virus ◽

Steady State ◽

Kinetic Parameters ◽

Steady State Kinetic ◽

State Kinetic

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Subsite structure of the endo-type chitin deacetylase from a Deuteromycete, Colletotrichum lindemuthianum: an investigation using steady-state kinetic analysis and MS

Biochemical Journal ◽

10.1042/bj20030204 ◽

2003 ◽

Vol 374 (2) ◽

pp. 369-380 ◽

Cited By ~ 47

Author(s):

Omid HEKMAT ◽

Ken TOKUYASU ◽

Stephen G. WITHERS

Keyword(s):

Steady State ◽

Kinetic Parameters ◽

Intrinsic Rate ◽

Degree Of Polymerization ◽

Colletotrichum Lindemuthianum ◽

Chitin Deacetylase ◽

Steady State Kinetic ◽

Intrinsic Rate Constant ◽

Hydrolysis Of ◽

State Kinetic

The endo-type chitin deacetylase (EC 3.5.1.41) from a Deuteromycete, Colletotrichum lindemuthianum (ATCC 56676), catalyses the hydrolysis of the acetamido group of GlcNAc (2-acetamido-2-deoxy-d-glucose) residues in chitin or chito-oligosaccharides with a degree of polymerization (n) equal to or greater than 2. The steady-state kinetic parameters for the initial deacetylation reactions of (GlcNAc)2–6 were determined using a direct, continuous spectrophotometric assay in combination with ESI-MS (electrospray ionization MS) analysis of the products. The dependence of the observed Km and kcat/Km on n suggests the presence of four enzyme subsites (−2, −1, 0 and +1) that interact with GlcNAc residues from the non-reducing end to the reducing end of the substrate. The turnover number (kcat, 7 s−1) is independent of n and represents the intrinsic rate constant (kint) for the hydrolysis of the acetamido group in subsite 0. The subsite affinities for the GlcNAc residues were calculated from the observed kcat/Km values (A−2, −11.0; A−1, −1.5; A0, −7.7; A+1, −12.5 kJ·mol−1). The increments in the subsite affinities due to the recognition of the acetamido groups were calculated [ΔΔG(N-acetyl)=3.3, 0, 4.0 and 0 kJ·mol−1 for subsites −2, −1, 0 and +1 respectively]. The steady-state kinetic parameters for the second deacetylation reaction of (GlcNAc)4 were also determined using (GlcNAcGlcNAcGlcNGlcNAc) as the substrate. The comparison of the experimental and theoretical values (calculated using the subsite affinities) suggests that the mono-deacetylated substrate binds strongly in a non-productive mode occupying all four subsites, thereby inhibiting the second deacetylation reaction.

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