Abstract

The Michaelis constant (K(m)) and V(mas) (E0k(cat)) values for two mutant sets of enzymes were studied from the viewpoint of their definition in a rapid equilibrium reaction model and in a steady state reaction model. The "AMP set enzyme" had a mutation at the AMP-binding site (Y95F, V67I, and V67I/L76V), and the "ATP set enzyme" had a mutation at a possible ATP-binding region (Y32F, Y34F, and Y32A/Y34A). Reaction rate constants obtained using steady state model analysis explained discrepancies found by the rapid equilibrium model analysis. (i) The unchanged number of bound AMPs for Y95F and the wild type despite the markedly increased K(m) values for AMP of the AMP set of enzymes was explained by alteration of the rate constants of the AMP step (k(+2), k(-2)) to retain the ratio k(+2)/k(-2). (ii) A 100 times weakened selectivity of ATP for Y34F in contrast to no marked changes in K(m) values for both ATP and AMP for the ATP set of enzymes was explained by the alteration of the rate constants of the ATP steps. A similar alteration of the K(m) and k(cat) values of these enzymes resulted from distinctive alterations of their rate constants. The pattern of alteration was highly suggestive. The most interesting finding was that the rate constants that decided the K(m) and k(cat) values were replaced by the mutation, and the simple relationships between K(m), k(cat), and the rate constants of K(m)1 = k(+1)/k(-1) and k(cat) = k(f) were not valid. The nature of the K(m) and k(cat) alterations was discussed.

Highlights

  • The Michaelis constant (Km) and Vmax (E0kcat) values for two mutant sets of enzymes were studied from the viewpoint of their definition in a rapid equilibrium reaction model and in a steady state reaction model

  • Reaction rate constants obtained using steady state model analysis explained discrepancies found by the rapid equilibrium model analysis. (i) The unchanged number of bound AMPs for Y95F and the wild type despite the markedly increased Km values for AMP of the AMP set of enzymes was explained by alteration of the rate constants of the AMP step (k؉2, k؊2) to retain the ratio k؉2/k؊2. (ii) A 100 times weakened selectivity of ATP for Y34F in contrast to no marked changes in Km values for both ATP and AMP for the ATP set of enzymes was explained by the alteration of the rate constants of the ATP steps

  • 1) For an AMP site mutant set (Y95F, V67I, and V67I/ L76V), Tyr-95 is replaced by Phe, because Tyr-95 has been assumed to participate in the binding of one of the substrates, and Leu-76 and Val-67 are near the active site Tyr-95

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Summary

EXPERIMENTAL PROCEDURES

Materials—The enzyme was ADK in porcine skeletal muscle obtained as a recombinant protein by expressing its cDNA. CD spectra in the 280 nm region of Y95F and the other mutant proteins lacking Tyr were less negative than for the wild type, presumably due to the substitution of Tyr by Phe. conformations of mutants had no effect due to the replacement of every target amino acid residue in the mutant enzymes. These reaction rates were further analyzed by using the random Bi Bi model with rapid equilibrium or steady state assumptions. 1 with four Michaelis constants and one Vmax, where SM and ST are the concentrations of AMP and ATP, respectively, and Vmax is equal to kcat [E]0. [E]0 is omitted by making [E]0 unity

STSM ϩ
TRANSFORMATION MATRIX A
RESULTS
ATP GTP CTP ITP UTP AMP CMP GMP IMP
DISCUSSION
ATP GTP CTP ITP UTP AMP CMP GMP IMP UMP
TABLE V Coefficients and their component rate parameters
Nucleotide concentration
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