A Kinetic and Isotope Effect Investigation of the Urease-Catalyzed Hydrolysis of Hydroxyurea

Abstract

The urease-catalyzed hydrolysis of hydroxyurea is known to exhibit biphasic kinetics, showing a rapid burst phase followed by a slow plateau phase. Kinetic isotope effects for both phases of this reaction were measured at pH 6.0 and 25°C. The observed nitrogen isotope effects for the ammonia leaving group were 15 (V/K) NH3 = 1.0016 ± 0.0005 during the burst phase and 15 (V/ K) NH3 = 1.0019 ± 0.0007 during the plateau phase while those for the hydroxylamine leaving group are 15 (V/K) NH2OH = 1.0013 ± 0.0005 for the burst phase and 15 (V/K) NH2OH = 1.0022 ± 0.0003 for the plateau phase. These isotope effects are consistent with a rate-determining step that occurs prior to breaking either of the two possible C-N bonds. The observed carbonyl carbon isotope effects are 13 (V/K) = 1.0135 ± 0.0003 during the burst phase and 13 (V/K) = 1.0178 ± 0.0003 during the plateau phase. The similarity of the magnitude of the carbon isotope effects argues for formation of a common intermediate during both phases.

Synthesis of Symmetric and Unsymmetric Secondary Amines from the Ligand-Promoted Ruthenium-Catalyzed Deaminative Coupling Reaction of Primary Amines

“…21 In a urease-catalyzed hydrolysis of hydroxyurea, Cleland and co-workers observed the significant carbon isotope effect on the carbonyl carbon but not on the nitrogen atom, which argues for the formation of a common intermediate prior to the C–N bond cleavage step. 22 …”

Mechanistic investigations of the hydrolysis of amides, oxoesters and thioesters via kinetic isotope effects and positional isotope exchange

“…In delta notation a superscript denotes the isotopes in question (e.g. 13 δ for the 13 C/ 12 C ratio; 34 δ is for the 34 S/ 32 S ratio). Published equations then allow calculation of the KIE from these measured delta values [3].…”

“…The KIE investigation of hydroxyurea hydrolysis was designed in a similar manner to that for semicarbazide [34]. It was hoped that hydroxyurea would offer an improvement over semicarbazide, namely elimination of the secondary KIE from the outer nitrogen of the \NHNH 2 leaving group.…”

“…The commitment factors (k 5 /k 4 ) for urea and its two derivatives can be calculated from the observed KIEs plus EIEs estimated from model reactions (see reference [34] for details). In this case the larger the commitment factor the more frequently the tetrahedral intermediate goes on to products (C\N bond breaking) versus returning to substrates.…”

Triple Isotopic Labeling and Kinetic Isotope Effects: Exposing H-Transfer Steps in Enzymatic Systems

“…Competitive experiments, while limited to measuring the isotope effect on the second order rate constant k cat /K M , (4) are not affected by different contaminants (since both isotopologues compete in the same reaction mixture) and can utilize the radioisotope tritium ( 3 H or T) in trace amounts, thus allowing examination of all three isotopes of hydrogen (H, D, and T, or 1 H, 2 H, and 3 H respectively). Heavy-atom KIEs can be accurately determined with the superior precision of competitive experiments (e.g., 15 N V/K effects of 1.0022 ± 0.0003 using an isotope ratio mass spectrometer (5), or 1.034 ± 002 using radioisotopes 14 C and 3 H (6)). The competitive method is thus the preferred choice for high-precision measurements of KIEs and is the only method to utilize radioistopes such as T in trace amounts.…”