Chymotrypsin Catalysis in the Presence of Formaldehyde. The pH-Dependence of Ester Hydrolysis

Abstract

1.The maximum steady state rate (kcat) of the a-chymotrypsin-catalyzed hydrolysis of acetyl-L-tyrosine ethyl ester a t 20" C in 0.15 M KC1 is 136 sec-l and is controlled by a group with p K 6.6. From a kJKL versus pH plot, two groups with pK' 6.6 and 8.3 are seen. I n 3.1 M formaldehyde, kcat is 16.3 sec-1 with the rate controlled by a group with p K 6.6 and the kCaJKm versus pH profile is bell-shaped with pK values of 6.4 and 8.4. 2.The active site concentration of a-chymotrypsin in water and in formaldehyde is identical. 3. The modification of chymotrypsin activity by formaldehyde is complete in less than 5 see and is completely reversible.4. Diisopropylphosphofluoridate completely inhibits a-chymotrypsin in the presence of formaldehyde although a t a slower rate. There is little or no inhibition with ~-1-tosylamido-2-phenylethyl chloromethyl ketone.5. These results lead to the conclusion that the active site has been modified with retention of catalytic activity. From a consideration of the reactivity of formaldehyde with the functional groups of the enzyme, it is suggested that the modification is the result of N-hydroxymethylation of the imidazole group(s) of a-chymotrypsin.A large body of evidence has accumulated which suggests that the mechanism of chymotrypsin catalysis proceeds via an acyl enzyme intermediate accord- During the course of some titration studies of chymotrypsin in formaldehyde, it was observed that chymotrypsin was enzymatically active in this solvent although less so than in water. It was also determined that all active sites were functional. A literature search revealed that other investigators [29,30] had also observed that formaldehyde inhibited chymotrypsin activity.

A combined spectroscopic and molecular modeling Study on structure-function-dynamics under chemical modification: Alpha-chymotrypsin with formalin preservative

“…Hence, whether formalin modification causes impairment of substrate binding at the active site cannot be discerned from Michaelis-Menten kinetics study of CHT under formalin modification. However, chymotrypsin goes through burst kinetics, we observed a longer initial phase for the formalin treated CHT ( Supplementary Figure S2 ), suggesting retardation in the de-acylation of Ser-195 due to residue modification upon formalin treatment, which was also echoed in previous literature ( Marini and Martin, 1971 ). Stability of the secondary and tertiary structure indicated to residue modification at the catalytic S1 pocket as the possible architect in reduction of catalytic activity of the enzyme in presence of formalin ( Figure 3 ), further any change to the mass of the enzyme due to crosslinking would have reflected in its hydrodynamic diameter, which we found no evidence of in our DLS study.…”

“…A reduction in the apparent catalytic efficiency (k cat /K m ) with increasing formalin concentration indicates a reduced catalytic activity which likely originates from a possible modification of the substrate binding site as indicated from previous reports. ( Marini and Martin, 1971 ; Martin et al, 1972 ). however, it did not escape our attention that the reduced catalytic activity could also be caused due to enzyme aggregation and precipitation due to formalin modification.…”

“…The Lineweaver-Burk plot ( Supplementary Figure S1 ) and Table 1 indicates formalin exhibiting competitive inhibition (no change in Vmax and increasing K m ) which may be inaccurate. Since formalin covalently modifies the active site residues causing irreversible covalent inhibition and can cause crosslinks ( Marini and Martin, 1971 ; Martin et al, 1972 ; Tayri-Wilk et al, 2020 ) understanding the kinetic parameters of CHT using Lineweaver-Burk plot (Michealis-Menten kinetics) may not be accurate. A crucial assumption of the Michaelis–Menten model is that the total enzyme concentration does not change over time.…”