Imine Deaminase Activity and Conformational Stability of UK114, the Mammalian Member of the Rid Protein Family Active in Amino Acid Metabolism

Reactive intermediate deaminase (Rid) protein family is a recently discovered group of enzymes that is conserved in all domains of life and is proposed to play a role in the detoxification of reactive enamines/imines. UK114, the mammalian member of RidA subfamily, was identified in the early 90s as a component of perchloric acid-soluble extracts from goat liver and exhibited immunomodulatory properties. Multiple activities were attributed to this protein, but its function is still unclear. This work addressed the question of whether UK114 is a Rid enzyme. Biochemical analyses demonstrated that UK114 hydrolyzes α-imino acids generated by l- or d-amino acid oxidases with a preference for those deriving from Ala > Leu = l-Met > l-Gln, whereas it was poorly active on l-Phe and l-His. Circular Dichroism (CD) analyses of UK114 conformational stability highlighted its remarkable resistance to thermal unfolding, even at high urea concentrations. The half-life of heat inactivation at 95 °C, measured from CD and activity data, was about 3.5 h. The unusual conformational stability of UK114 could be relevant in the frame of a future evaluation of its immunogenic properties. In conclusion, mammalian UK114 proteins are RidA enzymes that may play an important role in metabolism homeostasis also in these organisms.

. Determination of Vmax and Km for L-Leu (left) and L-Ala (right) for the reaction of LAAO as monitored from H2O2 production (v, H2O2; circles, left vertical axis) or semicarbazone formation from the imino acid (v, IA, squares, right vertical axis). The curves are the fit of the initial velocity data obtained by monitoring semicarbazone formation to the Michaelis Menten equation (v = Vmax*S/(Km + S), with: v, initial reaction velocity; Vmax, maximum velocity; Km, Michaelis-Menten constant). The initial velocity is expressed as mM H2O2 or semicarbazone formed per minute per mg LAAO. The steady-state kinetic parameters calculated for the reactions are summarized in Table S2.  (1) v The deiminase activity of UK114 is measured as the decrease of initial velocity of formation of semicarbazone from the imino acid produced by L-or D-amino acid oxidase (Supplementary Figure  S4). Thus, the system consists of two parallel competing pseudo-first order reactions: A → B, which occurs with an apparent rate constant kB, and A → C, which takes place with an apparent rate constant kc. A is the imino acid formed by L-or D-amino acid oxidase; B is the semicarbazone formed upon reaction of the imino acid with semicarbazide.
Since the initial velocity of the reaction is measured, semicarbazide concentration can be considered constant. Thus, the velocity of semicarbazone formation (vB = k1*[A]*[semicarbazide]) is a pseudo-first order reaction (vB = kB*[A], with kB = k1*[semicarbazide]). Also the UK114-catalysed hydrolysis of the imino acid (A) into the corresponding keto acid and ammonia is a pseudo-first order reaction with kC = k2*[UK114], with k2 = (kcat*[A])/(Km + [A]), i.e., the velocity predicted by the Michaelis-Menten equation with kcat and Km being the turnover number and the Km of UK114 with the given imino acid, respectively. Since the imino acid is generated in situ by the L-or D-amino acid reaction, at the same rate in all assays, and the initial velocities are measured, kC can also be considered constant.
For two parallel competing (pseudo)-first order reactions, the concentrations of B and C can be written as in [1]: Thus, at any given time: Since vC is the difference between the velocity measured in the absence of UK114 (v0) and the velocity of semicarbazone formation measured in its presence (vB), the expression becomes: , K50 is related to the specificity of UK114 for the different imino acids.
It should be noted that the curve relating the residual activity (in percent) and the UK114 concentration did not change when the initial velocity of formation of the imino acid (in the absence of UK114) was half that routinely used in these experiments. Thus, it appears that, under the assay conditions, UK114 works under kcat/Km conditions, i.e., its reaction velocity is linearly dependent upon substrate concentration and K50 relates to the catalytic efficiency of UK114 with the given imino acid.
The initial part of the curve relating residual activity to UK114 concentration is linear. By calculating the limit for [UK114] → 0 of the first derivative of the expression of v as a function of [UK114] one obtains the slope of the linear part of the curve, which corresponds to the apparent turnover number of UK114 with a given imino acid once it is multiplied by the initial velocity measured in the absence of UK114 expressed as µ M semicarbazone formed/min lim UK114→0 dv d [UK114] = − 100 K 50