Beef Heart Malic Dehydrogenases: VI. A pH-DEPENDENT TRANSITION OBSERVED IN THE PRESENCE OF REDUCED DIPHOSPHOPYRIDINE NUCLEOTIDE

Abstract

Abstract The constants for the dissociation of reduced diphosphopyridine nucleotide from both the binary (E. DPNH) and ternary (E . DPNH . d-malate) complexes of bovine heart supernatant malic dehydrogenase have been determined at pH values between 6 and 10 by spectrofluorometric techniques. The constants have minimum values at pH 6.5. Above pH 7, the presence of d-malate stabilizes bound DPNH. Furthermore, the dissociation constant of the binary complex increases at pH values above 9.0, although this is not observed for the ternary complex dissociation constant. The changes are interpreted as reflecting the titration of groups on the protein involved in the binding of DPNH. By use of similar techniques, the dissociation constant of the ternary complex with respect to D-malate has been measured. This constant is unchanged beween pH 6 and 8, but increases above pH 8. A group on the protein dissociating at pH 9.0 to 9.5 is considered to be involved in the binding of d-malate. Measurements of the quantum yields of fluorescence of DPNH in the binary and ternary complexes have been made between pH 6 and pH 10. No change in the quantum yield of the free protein can be observed in the pH range from 6 to 10. The results indicate that the quantum yield of DPNH in the complexes cannot be simply related to its dissociation constant. Some of the effect of d-malate on the DPNH quantum yield is attributed to local electronic interactions at the DPNH-binding site, producing changes in coenzyme fluorescence independent of changes in the stability of the bound DPNH. A parameter, q, which defines some properties of the energy transfer phenomenon is derived. In agreement with theoretical predictions, the energy transfer appears to be mainly nonradiative in nature. Examination of the q values indicates that a transition occurs between pH 7.0 and 7.5. A similar transition is observed with the ternary complex. The extent of quenching of the protein fluorescence upon binding of DPNH or DPNH + d-malate has also been measured. The degree of quenching, measured between pH 7.5 and 10.0, reflects the efficiency of energy transfer. In this pH region, the efficiency of energy transfer for both binary and ternary complexes decreases by 40 to 50% with increasing pH. The efficiency of energy transfer is about 10% greater in the ternary than in the binary complex. Below pH 7.5 the observed quenching is a reflection both of the energy transfer process and of quenching arising from other mechanisms. Optical rotatory dispersion studies of free supernatant malic dehydrogenase at pH 6.5 and 8.5, and of enzyme bound to DPNH at pH 8.5, gave almost identical b0 values. However, measurements carried out at pH 6.5 with the enzyme-DPNH complex show a 60% increase in b0 above the value obtained for this complex at pH 8.5. The pH- and DPNH-dependent transition observed from optical rotation and fluorescence studies may be related to a change in the kinetic properties of the enzyme (described in an earlier publication), which occurs over the same pH range.