Abstract
The primary structure of pyrophosphate-dependent phosphofructokinase (PFK) from Propionibacterium freudenreichii exhibits a low but significant level of sequence identity with Escherichia coli ATP-dependent PFK, permitting the tentative assignment of residues that may be involved in catalysis. Based on these assignments, the roles in catalysis of 2 aspartyl residues (Asp151 and Asp153) and 2 lysyl residues (Lys80 and Lys85) were examined. Mutagenesis of the Asp151 to alanine and serine reduced kcat by a factors of 2 x 10(4) and 4 x 10(5), respectively, while showing no change in Km for either substrate in the forward reaction or for metal ion in the back reaction. The kcat for Asp153 was decreased by a factor of 700 with no change in Km for pyrophosphate and an increase of about 20-fold in Km for fructose 6-P and close to 4-fold for magnesium ion. That these changes in the mutants were not the result of global conformational changes was indicated by their identical behavior during substrate-specific elution chromatography, ion-exchange chromatography, and limited proteolysis by trypsin and subtilisin. Mutations of Lys80 and Lys85 showed no significant changes in kinetic parameters, suggesting no involvement in mechanism or substrate binding. These and other results permit preliminary modeling of the active site of pyrophosphate-dependent PFK.
Highlights
From the Department of Biological Chemistry, University of Health Sciences, The Chicago Medical School, North Chicago, Illinois 60064
Assignments, the roles in catalysis of 2 aspartyl resi- The selection of appropriatecandidates for site-directed dLKaamlnuya,edesntesfaios4(nl)ArXeiwosepalneio'ntr"h"idene,esarrexnterhadssiumepnAbeebissncpratte'erc"iddavk).uteMelrcayeeniu,adnwdtcaLthtgih2oilelteynenbs.efsyyoThsliroahsrwkwoefecaasfiaictnrdthtdfguooenerrrssoeAoac(AsfcLhpts2a'yipsons3'X"neg'welooaatrionsn4fdormesozhyxnuomtetwcaenhg.nseeAimvtnoleetilhcsdyiaoislsfuftgmceuhradonistdehuidbebfiescAtPaabTtPniaoPtisi-ne-aPddlFsleytpKouefndfrnrioodeommseunrotPthfP.keFtfnrhmoKeewuafdPljreoeoPdnrmigrf-eeadEimeco.phficleiyiontlhhdoi aefe(sn7Amtb)TaeeonnePrnd-edecreased by a factorof 700 with no change inK, for dependent phosphofructokinases, a low but significant level pyrophosphate and an increase of abou20t -fold in K, of sequence identity with E. coli PFK has been established to for fructose6-P and close to 4-fold for magnesium ion. indicate thatthePPi-dependentandthe
Of about 23%was achieved (6).Despite this low level ofoverall similarity, a number of residues that have been implicated by x-ray crystallographic studies ( 7 ) of the ATP-dependent E. coli PFK to beinvolved in eitherFru 6-P binding or the phosphoryltransferase reaction have been tentatively aligned with residues in the PPi-dependent enzyme (6)
Summary
Purification of Wild-type and Mutant PFKs-Wild-type PPi-PFK and thfieve mutants, D151A, D151S, D153A, K90A, and K85A, were purified from bacterial extracts asdescribed under “Experimental Procedures.” Thisprocedure involves a very specific substrate elution of phosphocellulose that produces an enzyme that is neahrolymogeneous. Thisprocedure involves a very specific substrate elution of phosphocellulose that produces an enzyme that is neahrolymogeneous. Those mutants withvery low activity could notbequantitated accurately during the purificatpioroncedure by direct enzymic activityassay, because trace-contaminatingactivitiesthat contributed toa blank assay (withouPt Pi)interfered with the assay when it was necessaryto increase the amountof protein added to the cuvebttye 3-4 orders of magnitude. It should be noted that all enzymes were eluted from the P-cellulose columnby the same low concentration of the PFK reaction product, 0.5 m M Fru l,6-P2. These results suggest that the integrity of the sugar bisphosphate binding site has been retained in the mutants.
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