INTRASUBUNIT VS INTERSUBUNIT COMMUNICATION IN THE HOMODIMERIC RESTRICTION ENZYME ECORV - THR-37 AND LYS-38 INVOLVED IN INDIRECT READOUT AREONLY IMPORTANT FOR THE CATALYTIC ACTIVITY OF THEIR OWN SUBUNIT

EcoRV is a dimer of two identical subunits which together form one bin ding site for the double-stranded DNA substrate. Concerted cleavage of both strands of the duplex requires intersubunit communication to syn chronize the two catalytic centers of EcoRV. Here we address the quest ion of how contacts to the DNA backbone trigger conformational changes which lead to the activation of bath catalytic centers. The structure of the specific EcoRV-DNA complex shows that a region including amino acids Tn 37 and Lys 38 is involved in interactions with the DNA backb one and is a candidate for intersubunit communication. Homodimeric Eco RV T37A and K38A variants have a 1000-fold reduced catalytic activity. To examine whether Thr 37 and Lys 38 of one subunit affect the cataly tic center in the same subunit and/or in the other subunit, we have pr oduced heterodimeric variants containing a Thr 37 --> Ala or Lys 38 -- > Ala substitution in one subunit combined with a wild type (wt) subun it (wt/T37A and wt/ K38A) or with a subunit which contains an amino ac id substitution (Asp 90 --> Ala) in the active site (D90A/T37A and D90 A/K38A). Cleavage experiments with supercoiled pAT153 show that wt/T37 A and wt/K38A preferentially nick the DNA. A steady-state kinetic anal ysis of the cleavage of an oligodeoxynucleotide substrate shows that t he activity of wt/T37A and wt/K38A is half of that of wild type EcoRV, whereas D90A/T37A and D90A/K38A are almost inactive. These results de monstrate that Thr 37 and Lys 38 affect primarily the catalytic center in their own subunit and that both subunits of EcoRV can be activated independently of each other. We suggest that Thr 37 and Lys 38 contro l the catalytic activity of the active site in their own subunit by po sitioning alpha-helix B.