Dependence of the equilibrium constant for peptide bond hydrolysis on the P1 and P1' residues

Anthony Giletto, Purdue University

Abstract

The equilibrium constant of hydrolysis, K$\rm{\sbsp{hyd}{o}}$, was measured for peptide bonds as a function of the P$\sb1$ and P$\sb1\sp\prime$ residue respectively. The dependence of $\sb{\rm F}$K$\sbsp{\rm hyd}{\rm o}$ on the P$\sb1$and P$\sb1\sp\prime$ residue was measured in short peptide models and the dependence of $\sb{\rm R}$K$\rm{\sbsp{hyd}{o}}$ on the P$\sb1$ residue was measured at a single, specified site in a small protein. The model used to measure the dependence of $\sb{\rm F}$K$\rm{\sbsp{hyd}{o}}$ on the P$\sb1$ residue was: Fa-X-Phe-NH$\sb2$ $\sbsp{\gets}{\to}$ Fa-X-O$\sp-$ + $\sp+$H$\sb2$-Phe-NH$\sb2$, where Fa is furylacryloyl and X is, in descending order of $\sb{\rm F}$K$\sbsp{\rm hyd}{\rm o}$, Asp$\sp\circ$, Thr, Arg$\sp+$, Ser, Lys$\sp+$, His$\sp+$, Gly, Glu$\sp\circ$, Asn Met, Gln, Hse, Phe, Tyr, Ala, Val, His$\sp\circ$, Trp, Leu, Ile, Glu$\sp-$, or Asp$\sp-$. The range of these equilibrium constants is a factor of fourteen. The reactive site peptide bond of turkey ovomucoid third domain (OMTKY3) was the small protein site used to measure the dependence of $\sb{\rm R}$K$\sbsp{\rm hyd}{\rm o}$ on the P$\sb1$ residue. In descending order of $\sb{\rm R}$K$\sbsp{\rm hyd}{\rm o}$, substitutions at position 18 (P$\sb1$ residue) included: Val, Phe, Ile, Tyr, Trp, His$\sp+$, Ala, Ser, Gln, Asn, Arg$\sp+$, Leu (wild type), Glu$\sp-$, Lys$\sp+$, Thr, and Asp$\sp-$. The range of these equilibrium constants is a factor of five. Since the reactive site residues in proteinase inhibitors are exposed, a strong positive correlation was expected between $\sb{\rm R}$K$\sbsp{\rm hyd}{\rm o}$ of X$\sp{18}$-OMTKY3 and $\sb{\rm F}$K$\sbsp{\rm hyd}{\rm o}$ of Fa-X-Phe-NH$\sb2$, but it was not found. However, when $\sb{\rm R}$K$\sbsp{\rm hyd}{\rm o}$ values of native X$\sp{18}$-OMTKY3 were converted to that for thermally denatured X$\sp{18}$-OMTKY3 by the use of a thermodynamic cycle, a very strong positive correlation was seen with the dependence of $\sb{\rm F}$K$\sbsp{\rm hyd}{\rm o}$ on P$\sb1$ in Fa-X-Phe-NH$\sb2$. Cbz-Trp-X-NH$\sb2$ $\sbsp{\gets}{\to}$ Cbz-Trp-O$\sp-$ + $\sp+$H$\sb2$-X-NH$\sb2$ was the model used to measure the dependence of $\sb{\rm F}$K$\sbsp{\rm hyd}{\rm o}$ on the P$\sb1\sp\prime$ residue where Cbz is benzyloxycarbonyl and X is, in descending order of $\sb{\rm F}$K$\sbsp{\rm hyd}{\rm o}$, His$\sp\circ$, Ala, Asp$\sp-$, D-Ala, Glu$\sp-$, Ser, Gly, Val, Lys$\sp+$, Leu, Ile, Thr, Trp, Met, Gln, Asn, Arg$\sp+$, Phe, Glu$\sp\circ$, D-Leu, Asp$\sp\circ$, or His$\sp+$. The range of $\sb{\rm F}$K$\sbsp{\rm hyd}{\rm o}$'s in this set spans a factor of twenty three. The $\sb{\rm F}$K$\sbsp{\rm hyd}{\rm o}$ values for the diasteriomeric pair Cbz-Trp-Leu-NH$\sb2$ and Cbz-Trp-D-Leu-NH$\sb2$ allows for the evaluation of the equilibrium constant for the conversion of these compounds into one another. Acid ionization constants were measured and $\sb{\rm F}$K$\sbsp{\rm hyd}{\rm o}$'s were converted to $\sb{\rm F}$K$\sbsp{\rm hyd}{\rm class}$'s. This corrects for the dependence of $\sb{\rm F}$K$\sbsp{\rm hyd}{\rm o}$ on the pK$\sb{\rm a}$'s of the ionizable groups generated by hydrolysis. Following the $\sb{\rm F}$K$\sbsp{\rm hyd}{\rm o}$ to $\sb{\rm F}$K$\sbsp{\rm hyd}{\rm class}$ conversion, the range for the Fa-X-Phe-NH$\sb2$ series decreases significantly, while. the range for the Cbz-Trp-X-NH$\sb2$ increases. These results suggest that peptide bond strength is only slightly dependent on the P$\sb1$ residue but strongly dependent on the P$\sbsp{1}{\prime}$ residue.

Degree

Ph.D.

Advisors

Laskowski, Purdue University.

Subject Area

Biochemistry|Organic chemistry|Chemistry

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